Identification of DPP-IV inhibitory peptides derived from buffalo colostrum: Mining through bioinformatics, in silico and in vitro approaches.

Bioactive peptides derived from foods provide physiological health benefits beyond nutrition. This study focused on profiling small peptide inhibitors against two key serine proteases, dipeptidyl peptidase-IV (DPP-IV) and prolyl oligopeptidase (POP). DPP-IV is a well-known protein involved in diverse pathways regulating inflammation, renal, cardiovascular physiology, and glucose homeostasis. POP is yet another key target protein for neurodegenerative disorders. The study evaluated peptide libraries of buffalo colostrum whey and fat globule membrane proteins derived from pepsin and pepsin-pancreatin digestion through in silico web tools and structure-based analysis by molecular docking and binding free-energy estimation, followed by in vitro assay for DPP-IV inhibition for the lead peptides. The bioinformatic study indicated 49 peptides presented motifs with DPP-IV inhibition while 5 peptides with sequences for POP inhibition. In the molecular docking interactions study, 22 peptides interacted with active site residues of DPP-IV and 3 peptides with that of POP. The synthesized peptides, SFVSEVPEL and LTFQHNF inhibited DPP-IV in vitro with an IC50 of 193.5 µM and 1.782 mM, respectively. The study revealed the key residues for inhibition of DPP-IV and POP thus affirming the DPP-IV inhibitory potential of milk-derived peptides.

Production of the prolyl endoprotease (PEP) from Aspergillus sp. FSDE 16 by solid-state fermentation (SSF) and use for producing a gluten-free beer.

Beer is a beverage that contains gluten and cannot be consumed by people with celiac disease. In this context, the enzyme prolyl endoprotease (PEP) can be used to reduce the gluten content in beer. The present study aimed to produce the PEP from Aspergillus sp. FSDE 16 using solid-state fermentation with 5 conditions and comparing with a similar commercial enzyme produced from Aspergillus niger in the production of a gluten-free beer. The results of the performed cultures showed that during the culture, the most increased protease activity (54.46 U/mL) occurred on the 4th day. In contrast, for PEP, the highest activity (0.0356 U/mL) was obtained on the 3rd day of culture in condition. Regarding beer production, cell growth, pH, and total soluble solids showed similar behavior over the 7 days for beers produced without enzyme addition or with the addition of commercial enzyme and with the addition of the enzyme extract produced. The addition of the enzyme and the enzyme extract did not promote changes, and all the beers produced showed similar and satisfactory results, with acid pH between 4 and 5, total soluble solids ranging from 4.80 to 5.05, alcohol content ranging from 2.83% to 3.08%, and all beers having a dark character with deep amber and light copper color. Gluten removal was effectively using the commercial enzyme and the enzyme produced according to condition (v) reaching gluten concentrations equal to 17 ± 5.31 and 21.19 ± 11.28 ppm, respectively. In this way, the production of the enzyme by SSF and its application in the removal of gluten in beer was efficient.

Multiomics Analyses Identify Proline Endopeptidase-Like Protein As a Key Regulator of Protein Trafficking, a Pathway Underlying Alzheimer's Disease Pathogenesis.

Current treatments for Alzheimer's disease (AD) help reduce symptoms for a limited time but do not treat the underlying pathology. To identify potential therapeutic targets for AD, an integrative network analysis was previously carried out using 364 human postmortem control, mild cognitive impairment, and AD brains. This analysis identified proline endopeptidase-like protein (PREPL), an understudied protein, as a downregulated protein in late-onset AD patients. In this study we investigate the role of PREPL. Analyses of data from human postmortem samples and PREPL knockdown (KD) cells suggest that PREPL expression modulates pathways associated with protein trafficking, synaptic activities, and lipid metabolism. Furthermore, PREPL KD impairs cell proliferation and modulates the structure of vesicles, levels of neuropeptide-processing enzymes, and secretion of neuropeptides. In addition, decrease in PREPL levels leads to changes in the levels of a number of synaptic proteins as well as changes in the levels of secreted amyloid beta (Aß) 42 peptide and Tau phosphorylation. Finally, we report that local decrease in PREPL levels in mouse hippocampus attenuates long-term potentiation, suggesting a role in synaptic plasticity. Together, our results indicate that PREPL affects neuronal function by modulating protein trafficking and synaptic function, an important mechanism of AD pathogenesis. SIGNIFICANCE STATEMENT: Integrative network analysis reveals proline endopeptidase-like protein (PREPL) to be downregulated in human sporadic late-onset Alzheimer's disease brains. Down regulation of PREPL leads to increases in amyloid beta secretion, Tau phosphorylation, and decreases in protein trafficking and long-term potentiation.

Rational Design of a Highly Specific Prolyl Endopeptidase To Activate the Antihypertensive Effect of Peptides.

Enzymatic hydrolysis of food-derived proteins to produce bioactive peptides could activate food functions such as antihypertension. However, the diversity of enzymatic hydrolysis products can reduce bioactive peptides' efficacy. Highly specific proteases can homogenize the hydrolysis products to reduce the production of impotent peptides. In this study, we successfully obtained M. xanthus prolyl endopeptidase mutant Y451M by constraint/free molecular dynamics simulations and binding energy calculations. The specificity of Y451M for proline was increased by 286 % compared to WT, while its activity was almost unchanged. Milk-derived substrates processed with Y451M showed an antihypertensive effect that was 567 % higher than without enzymes. The ability to activate food antihypertension increased 152 % and the use of enzyme by 192 % compared with WT. Specific proteases are thus valuable tools in the processing of complex substrates to obtain bioactive peptides.

Prolyl oligopeptidase inhibition ameliorates experimental pulmonary fibrosis both in vivo and in vitro.

BACKGROUND: Pulmonary fibrosis is a progressive disease characterized by lung remodeling due to excessive deposition of extracellular matrix. Although the etiology remains unknown, aberrant angiogenesis and inflammation play an important role in the development of this pathology. In this context, recent scientific research has identified new molecules involved in angiogenesis and inflammation, such as the prolyl oligopeptidase (PREP), a proteolytic enzyme belonging to the serine protease family, linked to the pathology of many lung diseases such as pulmonary fibrosis. Therefore, the aim of this study was to investigate the effect of a selective inhibitor of PREP, known as KYP-2047, in an in vitro and in an in vivo model of pulmonary fibrosis. METHODS: The in vitro model was performed using human alveolar A549 cells. Cells were exposed to lipopolysaccharide (LPS) 10 µg/ml and then, cells were treated with KYP-2047 at the concentrations of 1 µM, 10 µM and 50 µM. Cell viability was evaluated by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium (MTT) bromide colorimetric assay, while inflammatory protein expression was assessed by western blots analysis. The in vivo model was induced in mice by intra-tracheal administration of bleomycin (1 mg/kg) and then treated intraperitoneally with KYP-2047 at doses of 1, 2.5 and 5 mg/kg once daily for 12 days and then mice were sacrificed, and lung tissues were collected for analyses. RESULTS: The in vitro results demonstrated that KYP-2047 preserved cell viability, reduced inflammatory process by decreasing IL-18 and TNF-α, and modulated lipid peroxidation as well as nitrosative stress. The in vivo pulmonary fibrosis has demonstrated that KYP-2047 was able to restore histological alterations reducing lung injury. Our data demonstrated that KYP-2047 significantly reduced angiogenesis process and the fibrotic damage modulating the expression of fibrotic markers. Furthermore, KYP-2047 treatment modulated the IκBα/NF-κB pathway and reduced the expression of related pro-inflammatory enzymes and cytokines. Moreover, KYP-2047 was able to modulate the JAK2/STAT3 pathway, highly involved in pulmonary fibrosis. CONCLUSION: In conclusion, this study demonstrated the involvement of PREP in the pathogenesis of pulmonary fibrosis and that its inhibition by KYP-2047 has a protective role in lung injury induced by BLM, suggesting PREP as a potential target therapy for pulmonary fibrosis. These results speculate the potential protective mechanism of KYP-2047 through the modulation of JAK2/STAT3 and NF-κB pathways.

High-level production of Aspergillus niger prolyl endopeptidase from agricultural residue and its application in beer brewing.

BACKGROUND: Prolyl endopeptidase from Aspergillus niger (AN-PEP) is a prominent serine proteinase with various potential applications in the food and pharmaceutical industries. However, the availability of efficient and low-cost AN-PEP remains a challenge owing to its low yield and high fermentation cost. RESULTS: Here, AN-PEP was recombinantly expressed in Trichoderma reesei (rAN-PEP) under the control of the cbh1 promoter and its secretion signal. After 4 days of shaking flask cultivation with the model cellulose Avicel PH101 as the sole carbon source, the extracellular prolyl endopeptidase activity reached up to 16.148 U/mL, which is the highest titer reported to date and the secretion of the enzyme is faster in T. reesei than in other eukaryotic expression systems including A. niger and Komagataella phaffii. Most importantly, when cultivated on the low-cost agricultural residue corn cob, the recombinant strain was found to secret a remarkable amount of rAN-PEP (37.125 U/mL) that is twice the activity under the pure cellulose condition. Furthermore, treatment with rAN-PEP during beer brewing lowered the content of gluten below the ELISA kit detection limit (< 10 mg/kg) and thereby, reduced turbidity, which would be beneficial for improving the non-biological stability of beer. CONCLUSION: Our research provides a promising approach for industrial production of AN-PEP and other enzymes (proteins) from renewable lignocellulosic biomass, which provides a new idea with relevant researchers for the utilization of agricultural residues.

Myrsinane-Type Diterpenes: A Comprehensive Review on Structural Diversity, Chemistry and Biological Activities.

Euphorbia species are important sources of polycyclic and macrocyclic diterpenes, which have been the focus of natural-product-based drug research due to their relevant biological properties, including anticancer, multidrug resistance reversal, antiviral, and anti-inflammatory activities. Premyrsinane, cyclomyrsinane, and myrsinane diterpenes are generally and collectively designated as myrsinane-type diterpenes. These compounds are derived from the macrocyclic lathyrane structure and are characterized by having highly oxygenated rearranged polycyclic systems. This review aims to describe and summarize the distribution and diversity of 220 myrsinane-type diterpenes isolated in the last four decades from about 20 Euphorbia species. Some myrsinane diterpenes obtained from Jatropha curcas are also described. Discussion on their plausible biosynthetic pathways is presented, as well as isolation procedures and structural elucidation using nuclear magnetic resonance spectroscopy. Furthermore, the most important biological activities are highlighted, which include cytotoxic and immunomodulatory activities, the modulation of efflux pumps, the neuroprotective effects, and the inhibition of enzymes such as urease, HIV-1 reverse transcriptase, and prolyl endopeptidase, among other biological effects.

Synergistic Pulmonoprotective Effect of Natural Prolyl Oligopeptidase Inhibitors in In Vitro and In Vivo Models of Acute Respiratory Distress Syndrome.

Acute respiratory distress syndrome (ARDS) is a highly morbid inflammatory lung disease with limited pharmacological interventions. The present study aims to evaluate and compare the potential pulmonoprotective effects of natural prolyl oligopeptidase (POP) inhibitors namely rosmarinic acid (RA), chicoric acid (CA), epigallocatechin-3-gallate (EGCG) and gallic acid (GA), against lipopolysaccharide (LPS)-induced ARDS. Cell viability and expression of pro-inflammatory mediators were measured in RAW264.7 cells and in primary murine lung epithelial and bone marrow cells. Nitric oxide (NO) production was also assessed in unstimulated and LPS-stimulated RAW264.7 cells. For subsequent in vivo experiments, the two natural products (NPs) with the most favorable effects, RA and GA, were selected. Protein, cell content and lipid peroxidation levels in bronchoalveolar lavage fluid (BALF), as well as histopathological changes and respiratory parameters were evaluated in LPS-challenged mice. Expression of key mediators involved in ARDS pathophysiology was detected by Western blotting. RA and GA favorably reduced gene expression of pro-inflammatory mediators in vitro, while GA decreased NO production in macrophages. In LPS-challenged mice, RA and GA co-administration improved respiratory parameters, reduced cell and protein content and malondialdehyde (MDA) levels in BALF, decreased vascular cell adhesion molecule-1 (VCAM-1) and the inducible nitric oxide synthase (iNOS) protein expression, activated anti-apoptotic mechanisms and down-regulated POP in the lung. Conclusively, these synergistic pulmonoprotective effects of RA and GA co-administration could render them a promising prophylactic/therapeutic pharmacological intervention against ARDS.

Inhibition of Prolyl Oligopeptidase Restores Prohibitin 2 Levels in Psychosis Models: Relationship to Cognitive Deficits in Schizophrenia.

Cognitive impairment represents one of the core features of schizophrenia. Prolyl Oligopeptidase (POP) inhibition is an emerging strategy for compensating cognitive deficits in hypoglutamatergic states such as schizophrenia, although little is known about how POP inhibitors exert their pharmacological activity. The mitochondrial and nuclear protein Prohibitin 2 (PHB2) could be dysregulated in schizophrenia. However, altered PHB2 levels in schizophrenia linked to N-methyl-D-aspartate receptor (NMDAR) activity and cognitive deficits are still unknown. To shed light on this, we measured the PHB2 levels by immunoblot in a postmortem dorsolateral prefrontal cortex (DLPFC) of schizophrenia subjects, in the frontal pole of mice treated with the NMDAR antagonists phencyclidine and dizocilpine, and in rat cortical astrocytes and neurons treated with dizocilpine. Mice and cells were treated in combination with the POP inhibitor IPR19. The PHB2 levels were also analyzed by immunocytochemistry in rat neurons. The PHB2 levels increased in DLPFC in cases of chronic schizophrenia and were associated with cognitive impairments. NMDAR antagonists increased PHB2 levels in the frontal pole of mice and in rat astrocytes and neurons. High levels of PHB2 were found in the nucleus and cytoplasm of neurons upon NMDAR inhibition. IPR19 restored PHB2 levels in the acute NMDAR inhibition. These results show that IPR19 restores the upregulation of PHB2 in an acute NMDAR hypoactivity stage suggesting that the modulation of PHB2 could compensate NMDAR-dependent cognitive impairments in schizophrenia.

Crystal structure and substrate recognition mechanism of the prolyl endoprotease PEP from Aspergillus niger.

Proteases are enzymes that are not only essential for life but also industrially important. Understanding the substrate recognition mechanisms of proteases is important to enhance the use of proteases. The fungus Aspergillus produces a wide variety of proteases, including PEP, which is a prolyl endoprotease from A. niger. Although PEP exhibits amino acid sequence similarity to the serine peptidase family S28 proteins (PRCP and DPP7) that recognize Pro-X bonds in the terminal regions of peptides, PEP recognizes Pro-X bonds not only in peptides but also in proteins. To reveal the structural basis of the prolyl endoprotease activity of PEP, we determined the structure of PEP by X-ray crystallography at a resolution of 1.75 Å. The PEP structure shows that PEP has a wide-open catalytic pocket compared to its homologs. The characteristic catalytic pocket structure of PEP is predicted to be important for the recognition of protein substrates.

Diversification of phenolic glucosides by two UDP-glucosyltransferases featuring complementary regioselectivity.

BACKGROUND: Glucoside natural products have been showing great medicinal values and potentials. However, the production of glucosides by plant extraction, chemical synthesis, and traditional biotransformation is insufficient to meet the fast-growing pharmaceutical demands. Microbial synthetic biology offers promising strategies for synthesis and diversification of plant glycosides. RESULTS: In this study, the two efficient UDP-glucosyltransferases (UGTs) (UGT85A1 and RrUGT3) of plant origin, that are capable of recognizing phenolic aglycons, are characterized in vitro. The two UGTs show complementary regioselectivity towards the alcoholic and phenolic hydroxyl groups on phenolic substrates. By combining a developed alkylphenol bio-oxidation system and these UGTs, twenty-four phenolic glucosides are enzymatically synthesized from readily accessible alkylphenol substrates. Based on the bio-oxidation and glycosylation systems, a number of microbial cell factories are constructed and applied to biotransformation, giving rise to a variety of plant and plant-like O-glucosides. Remarkably, several unnatural O-glucosides prepared by the two UGTs demonstrate better prolyl endopeptidase inhibitory and/or anti-inflammatory activities than those of the clinically used glucosidic drugs including gastrodin, salidroside and helicid. Furthermore, the two UGTs are also able to catalyze the formation of N- and S-glucosidic bonds to produce N- and S-glucosides. CONCLUSIONS: Two highly efficient UGTs, UGT85A1 and RrUGT3, with distinct regioselectivity were characterized in this study. A group of plant and plant-like glucosides were efficiently synthesized by cell-based biotransformation using a developed alkylphenol bio-oxidation system and these two UGTs. Many of the O-glucosides exhibited better PEP inhibitory or anti-inflammatory activities than plant-origin glucoside drugs, showing significant potentials for new glucosidic drug development.

Inhibition-mediated changes in prolyl oligopeptidase dynamics possibly related to α-synuclein aggregation.

The formation of protein aggregates is one of the leading causes of neuronal malfunction and subsequent brain damage in many neurodegenerative diseases. In Parkinson's disease, α-synucleins are involved in the accumulation of aggregates. The origin of aggregation is unknown, but there is convincing evidence that it can be reduced by prolyl oligopeptidase (PREP) inhibition. This effect cannot simply be related to the inhibition of the enzyme's catalytic function since not all PREP inhibitors stop α-synuclein aggregation. Finding differences in the dynamics of the enzyme inhibited by different compounds would allow us to identify the protein regions involved in the interaction between PREP and α-synuclein. Here, we investigate the effects of three PREP inhibitors, each of which affects α-synuclein aggregation to a different extent. We use molecular dynamics modelling to identify the molecular mechanisms underlying PREP inhibition and find structural differences between inhibitor-PREP systems. We suggest that even subtle variations in enzyme dynamics affect its interactions with α-synucleins. Our identification of these regions may therefore be biologically relevant in preventing α-synuclein aggregate formation.

The Role of Tß4-POP-Ac-SDKP Axis in Organ Fibrosis.

Fibrosis is a pathological process in which parenchymal cells are necrotic and excess extracellular matrix (ECM) is accumulated due to dysregulation of tissue injury repair. Thymosin ß4 (Tß4) is a 43 amino acid multifunctional polypeptide that is involved in wound healing. Prolyl oligopeptidase (POP) is the main enzyme that hydrolyzes Tß4 to produce its derivative N-acetyl-seryl-aspartyl-lysyl-proline (Ac-SDKP) which is found to play a role in the regulation of fibrosis. Accumulating evidence suggests that the Tß4-POP-Ac-SDKP axis widely exists in various tissues and organs including the liver, kidney, heart, and lung, and participates in the process of fibrogenesis. Herein, we aim to elucidate the role of Tß4-POP-Ac-SDKP axis in hepatic fibrosis, renal fibrosis, cardiac fibrosis, and pulmonary fibrosis, as well as the underlying mechanisms. Based on this, we attempted to provide novel therapeutic strategies for the regulation of tissue damage repair and anti-fibrosis therapy. The Tß4-POP-Ac-SDKP axis exerts protective effects against organ fibrosis. It is promising that appropriate dosing regimens that rely on this axis could serve as a new therapeutic strategy for alleviating organ fibrosis in the early and late stages.

Prolyl oligopeptidase inhibition reduces alpha-synuclein aggregation in a cellular model of multiple system atrophy.

Multiple system atrophy (MSA) is a fatal neurodegenerative disease where the histopathological hallmark is glial cytoplasmic inclusions in oligodendrocytes, rich of aggregated alpha-synuclein (aSyn). Therefore, therapies targeting aSyn aggregation and toxicity have been studied as a possible disease-modifying therapy for MSA. Our earlier studies show that inhibition of prolyl oligopeptidase (PREP) with KYP-2047 reduces aSyn aggregates in several models. Here, we tested the effects of KYP-2047 on a MSA cellular models, using rat OLN-AS7 and human MO3.13 oligodendrocyte cells. As translocation of p25α to cell cytosol has been identified as an inducer of aSyn aggregation in MSA models, the cells were transiently transfected with p25α. Similar to earlier studies, p25α increased aSyn phosphorylation and aggregation, and caused tubulin retraction and impaired autophagy in OLN-AS7 cells. In both cellular models, p25α transfection increased significantly aSyn mRNA levels and also increased the levels of inactive protein phosphatase 2A (PP2A). However, aSyn or p25α did not cause any cellular death in MO3.13 cells, questioning their use as a MSA model. Simultaneous administration of 10 µM KYP-2047 improved cell viability, decreased insoluble phosphorylated aSyn and normalized autophagy in OLN-AS7 cells but similar impact was not seen in MO3.13 cells.

Mycobacterium tuberculosis puromycin hydrolase displays a prolyl oligopeptidase fold and an acyl aminopeptidase activity.

Puromycin-hydrolizing peptidases have been described as members of the prolyl oligopeptidase peptidase family. These enzymes are present across all domains of life but still little is known of the homologs found in the pathogenic bacterium Mycobacterium tuberculosis. The crystal structure of a M. tuberculosis puromycin hydrolase peptidase has been determined at 3 Angstrom resolution, revealing a conserved prolyl oligopeptidase fold, defined by α/ß-hydrolase and ß-propeller domains with two distinctive loops that occlude access of large substrates to the active site. The enzyme displayed amino peptidase activity with a substrate specificity preference for hydrophobic residues in the decreasing order of phenylalanine, leucine, alanine and proline. The enzyme's active site is lined by residues Glu564 for the coordination of the substrates amino terminal moiety and His561, Val608, Tyr78, Trp306, Phe563 and Ty567 for the accommodation of hydrophobic substrates. The availability of a crystal structure for puromycin hydrolase of M. tuberculosis shall facilitate the development of inhibitors with therapeutic applications.

Prolyl oligopeptidase participates in the cytosine arabinoside-induced nuclear translocation of glyceraldehyde 3-phosphate dehydrogenase in a human neuroblastoma cell line.

Previously, we reported that glyceraldehyde 3-phosphate dehydrogenase (GAPDH) is a binding partner of prolyl oligopeptidase (POP) in neuroblastoma NB-1 cells and that the POP inhibitor, SUAM-14746, inhibits cytosine arabinoside (Ara-C)-induced nuclear translocation of GAPDH and protects against Ara-C cytotoxicity. To carry out a more in-depth analysis of the interaction between POP and GAPDH, we generated POP-KO NB-1 cells and compared the nuclear translocation of GAPDH after Ara-C with or without SUAM-14746 treatment to wild-type NB-1 cells by western blotting and fluorescence immunostaining. Ara-C did not induce the nuclear translocation of GAPDH and SUAM-14746 did not protect against Ara-C cytotoxicity in POP-KO cells. These results indicate that the anticancer effects of Ara-C not only include the commonly known antimetabolic effects, but also the induction of cell death by nuclear transfer of GAPDH through interaction with POP.

Computational design of substrate selective inhibition.

Most enzymes act on more than a single substrate. There is frequently a need to block the production of a single pathogenic outcome of enzymatic activity on a substrate but to avoid blocking others of its catalytic actions. Full blocking might cause severe side effects because some products of that catalysis may be vital. Substrate selectivity is required but not possible to achieve by blocking the catalytic residues of an enzyme. That is the basis of the need for "Substrate Selective Inhibitors" (SSI), and there are several molecules characterized as SSI. However, none have yet been designed or discovered by computational methods. We demonstrate a computational approach to the discovery of Substrate Selective Inhibitors for one enzyme, Prolyl Oligopeptidase (POP) (E.C 3.4.21.26), a serine protease which cleaves small peptides between Pro and other amino acids. Among those are Thyrotropin Releasing Hormone (TRH) and Angiotensin-III (Ang-III), differing in both their binding (Km) and in turnover (kcat). We used our in-house "Iterative Stochastic Elimination" (ISE) algorithm and the structure-based "Pharmacophore" approach to construct two models for identifying SSI of POP. A dataset of ~1.8 million commercially available molecules was initially reduced to less than 12,000 which were screened by these models to a final set of 20 molecules which were sent for experimental validation (five random molecules were tested for comparison). Two molecules out of these 20, one with a high score in the ISE model, the other successful in the pharmacophore model, were confirmed by in vitro measurements. One is a competitive inhibitor of Ang-III (increases its Km), but non-competitive towards TRH (decreases its Vmax).

Long-Term Social Isolation Reduces Expression of the BDNF Precursor and Prolyl Endopeptidase in the Rat Brain.

Early-life stress is a risk factor for the development of behavioral and cognitive disorders in humans and animals. Such stressful situations include social isolation in early postnatal ontogenesis. Behavioral and cognitive impairments associated with neuroplastic changes in brain structures. We have found that after ten weeks of social isolation, male Wistar rats show behavioral abnormalities and cognitive deficit, accompanied by an increase in the relative expression of gene encoding serine protease prolyl endopeptidase (PREP, EC 3.4.21.26) in the brain frontal cortex. The present study aimed to assess synaptophysin (SYP), brain-derived neurotrophic factor precursor (proBDNF), and PREP expression using Western blot in the brain structures - the hippocampus, frontal cortex, and striatum of the rats subjected to prolonged social isolation compared with group-housed animals. Twenty Wistar rats were used for this study (10 males and 10 females). Experimental animals (5 males and 5 females) were kept one per cage for nine months, starting from the age of one month. Ten-month-old socially isolated rats showed memory deficit in passive avoidance paradigm and Morris Water Maze and reactivity to novelty reduction. We used monoclonal antibodies for the Western blot analysis of the expression of SYP, proBDNF, and PREP in the rat brain structures. Social isolation caused a proBDNF expression reduction in the frontal cortex in females and a reduction in PREP expression in the striatum in males. These data suppose that neurotrophic factors and PREP are involved in the mechanisms of behavioral and cognitive impairments observed in the rats subjected to prolonged social isolation with an early life onset.

Prolyl Oligopeptidase Regulates Dopamine Transporter Oligomerization and Phosphorylation in a PKC- and ERK-Independent Manner.

Prolyl oligopeptidase (PREP) is a serine protease that binds to alpha-synuclein (aSyn) and induces its aggregation. PREP inhibitors have been shown to have beneficial effects in Parkinson's disease models by enhancing the clearance of aSyn aggregates and modulating striatal dopamine. Additionally, we have shown that PREP regulates phosphorylation and internalization of dopamine transporter (DAT) in mice. In this study, we clarified the mechanism behind this by using HEK-293 and PREP knock-out HEK-293 cells with DAT transfection. We tested the effects of PREP, PREP inhibition, and alpha-synuclein on PREP-related DAT regulation by using Western blot analysis and a dopamine uptake assay, and characterized the impact of PREP on protein kinase C (PKC) and extracellular signal-regulated kinase (ERK) by using PKC assay and Western blot, respectively, as these kinases regulate DAT phosphorylation. Our results confirmed our previous findings that a lack of PREP can increase phosphorylation and internalization of DAT and decrease uptake of dopamine. PREP inhibition had a variable impact on phosphorylation of ERK dependent on the metabolic state of cells, but did not have an effect on phosphorylation or function of DAT. PREP modifications did not affect PKC activity either. Additionally, a lack of PREP elevated a DAT oligomerization that is associated with intracellular trafficking of DAT. Our results suggest that PREP-mediated phosphorylation, oligomerization, and internalization of DAT is not dependent on PKC or ERK.

The Protective Role of Prolyl Oligopeptidase (POP) Inhibition in Kidney Injury Induced by Renal Ischemia-Reperfusion.

Ischemia/reperfusion injury (IRI) is a complex pathophysiological process characterized by blood circulation disorder caused by various factors, such as traumatic shock, surgery, organ transplantation, and thrombus. Severe metabolic dysregulation and tissue structure destruction are observed upon restoration of blood flow to the ischemic tissue. The kidney is a highly perfused organ, sensitive to ischemia and reperfusion injury, and the incidence of renal IRI has high morbidity and mortality. Several studies showed that infiltration of inflammatory cells, apoptosis, and angiogenesis are important mechanisms involved in renal IRI. Despite advances in research, effective therapies for renal IRI are lacking. Recently it has been demonstrated the role of KYP2047, a selective inhibitor of prolyl oligopeptidase (POP), in the regulation of inflammation, apoptosis, and angiogenesis. Thus, this research focused on the role of POP in kidney ischemia/reperfusion (KI/R). An in vivo model of KI/R was performed and mice were subjected to KYP2047 treatment (intraperitoneal, 0.5, 1 and 5 mg/kg). Histological analysis, Masson's trichrome and periodic acid shift (PAS) staining, immunohistochemical and Western blots analysis, real-time PCR (RT-PCR) and ELISA were performed on kidney samples. Moreover, serum creatinine and blood urea nitrogen (BUN) were quantified. POP-inhibition by KYP2047 treatment, only at the doses of 1 and 5 mg/kg, significantly reduced renal injury and collagen amount, regulated inflammation through canonical and non-canonical NF-κB pathway, and restored renal function. Moreover, KYP2047 modulated angiogenesis markers, such as TGF-ß and VEGF, also slowing down apoptosis. Interestingly, treatment with KYP2047 modulated PP2A activity. Thus, these findings clarified the role of POP inhibition in AKI, also offering novel therapeutic target for renal injury after KI/R.