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Congenital myasthenic syndrome-22 (CMS22, OMIM 616224) is a rare genetic disorder caused by deleterious genetic variation in the prolyl endopeptidase-like (PREPL) gene. Previous reports have described patients with deletions and nonsense variants in PREPL, but nothing is known about the effect of missense variants in the pathology of CMS22. In this study, we have functionally characterized missense variants in PREPL from 3 patients with CMS22, all with hallmark phenotypes. Biochemical evaluation revealed that these missense variants do not impair hydrolase activity, thereby challenging the conventional diagnostic criteria and disease mechanism. Structural analysis showed that the variants affect regions most likely involved in intraprotein or protein-protein interactions. Indeed, binding to a selected group of known interactors was differentially reduced for the 3 variants. The importance of nonhydrolytic functions of PREPL was investigated in catalytically inactive PREPL p.Ser559Ala cell lines, which showed that hydrolytic activity of PREPL is needed for normal mitochondrial function but not for regulating AP1-mediated transport in the transgolgi network. In conclusion, these studies showed that CMS22 can be caused not only by deletion and truncation of PREPL but also by missense variants that do not necessarily result in a loss of hydrolytic activity of PREPL.

BACKGROUND X-PROLYL AMINOPEPTIDASE 3: (XPNPEP3) mutations are known to cause nephronophthisis-like nephropathy-1 (NPHPL1), a rare autosomal-recessive kidney disease characterized by progressive kidney failure and cystic kidney disease in childhood. The full phenotypic spectrum associated with mutations in XPNPEP3 is not fully elucidated. CASE PRESENTATION: A 13-year-old Chinese female patient with intellectual disability presented with a 2-year history of convulsions and fatigue, with a recent episode of swelling, breathlessness, and nocturnal dyspnea lasting 10 days. The patient was diagnosed with heart failure and kidney failure. Whole exome sequencing revealed a homozygous c.970-2 A > G mutation in XPNPEP3 associated with severe cardiac dysfunction and neurological symptoms, including epilepsy and intellectual disability. Notably, kidney ultrasound did not reveal the typical changes of NPHPL1, and kidney failure was hypothesized to be secondary to cardiac dysfunction rather than primary kidney pathology. CONCLUSIONS: This case suggests the possible association of additional phenotypic features associated with XPNPEP3 mutations, emphasizing the need for further investigation into the heterogeneous clinical presentations associated with XPNPEP3 mutations. The findings highlight the importance of considering alternative phenotypes in patients with genetic mutations traditionally associated with specific diseases. Segregation and functional analyses are necessary to determine causality between the c.970-2 A > G XPNPEP3 mutation and disease., (© 2024. The Author(s).)

Background and Objectives: The main treatment for celiac disease is a gluten-free diet. Gluten infection occurs in 55%-32% of these patients, which can cause symptoms in these patients. Many patients with celiac disease are dissatisfied with a gluten-free diet and are interested in a non-dietary alternative treatment. Methods: In this study, probiotic samples of local dairy (milk, yogurt, and cheese) from Iran were collected in sterile conditions and after enzymatic extraction using a lysis buffer and a sonication method for enzyme activity of prolyl endopeptidase and aminopeptidase N were evaluated. Results: The highest absorption rate for Z-Gly-Pro-4-nitroanilide substrate that performs the evaluation of prolyl endopeptidase enzyme, respectively, for strains C10-1 (L. pentosus), B6-1 (L. fermentum), C5-2 (L.paracasei), Y5 (L.paracasei) and C11-1 (L. pentosus) and in relation to Leu-p-NA which assesses aminopeptidase N The highest uptake was observed for strains K1 (L. helveticus), B8-2 (L. plantarum), Y5 (L.paracasei), P2-3 and P35 (L. plantarum), respectively. All of the above strains showed significantly different uptake than the negative control containing buffer and substrate (P

Prolyl oligopeptidases (POPs) are atypical serine proteases that are unique in their involvement in the maturation and degradation of prolyl‐containing peptide hormones and neuropeptides. They are potential pharmaceutical targets for the treatment of several neurodegenerative disorders, such as Alzheimer's disease. In this study, the catalytic and substrate‐regulatory mechanisms of a novel bacterial POP from Microbulbifer arenaceous (MaPOP) were investigated. The crystal structure revealed that the catalytic triad of MaPOP was covered by the central tunnel of an unusual β‐propeller domain. The tunnel not only provided the sole access to the active site for oligopeptides, but also protected large structured peptides or proteins from accidental proteolysis. The enzyme was able to cleave angiotensin I specifically at the carboxyl side of the internal proline residue, but could not hydrolyze long‐chain bovine insulin B in vitro. Like the ligand‐free structure, MaPOP bound to the transition‐state analog inhibitor ZPR was also in a closed state, which was not modulated by the common 'latching loop' found in other POPs. The substrate‐assisted catalytic mechanism of MaPOP reported here may represent a common mechanism for all POPs. These results may facilitate a better understanding of the catalytic behavior of POPs under physiological conditions. [ABSTRACT FROM AUTHOR]

Prolyl oligopeptidase (POP) is a compelling therapeutic target associated with aging and neurodegenerative disorders due to its pivotal role in neuropeptide processing. Despite initial promise demonstrated by early-stage POP inhibitors, their progress in clinical trials has been halted at Phase I or II. This impediment has prompted the pursuit of novel inhibitors. The current study seeks to contribute to the identification of efficacious POP inhibitors through the design, synthesis, and comprehensive evaluation (both in vitro and in silico) of thiazolyl thiourea derivatives (5a-r). In vitro experimentation exhibited that the compounds displayed significant higher potency as POP inhibitors. Compound 5e demonstrated an IC 50 value of 16.47 ± 0.54 μM, representing a remarkable potency. A meticulous examination of the structure-activity relationship indicated that halogen and methoxy substituents were the most efficacious. In silico investigations delved into induced fit docking, pharmacokinetics, and molecular dynamics simulations to elucidate the intricate interactions, orientation, and conformational changes of these compounds within the active site of the enzyme. Moreover, our pharmacokinetic assessments confirmed that the majority of the synthesized compounds possess attributes conducive to potential drug development., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 Elsevier B.V. All rights reserved.)

We comment here on the article by Stefanolo et al entitled "Effect of Aspergillus niger prolyl endopeptidase in patients with celiac disease on a long-term gluten-free diet", published in the World Journal of Gastroenterology . Celiac disease is a well-recognized systemic autoimmune disorder. In genetically susceptible people, the most evident damage is located in the small intestine, and is caused and worsened by the ingestion of gluten. For that reason, celiac patients adopt a gluten-free diet (GFD), but it has some limitations, and it does not prevent re-exposure to gluten. Research aims to develop adjuvant therapies, and one of the most studied alternatives is supplementation with Aspergillus niger prolyl endopeptidase protease (AN-PEP), which is able to degrade gluten in the stomach, reducing its concentration in the small intestine. The study found a high adherence to the GFD, but did not address AN-PEP as a gluten immunogenic peptide reducer, as it was only tested in patients following a GFD and not in gluten-exposing conditions. This study opens up new research perspectives in this area and shows that further study is needed to clarify the points that are still in doubt., Competing Interests: Conflict-of-interest statement: The authors declare having no conflicts of interest., (©The Author(s) 2024. Published by Baishideng Publishing Group Inc. All rights reserved.)

Proteolytic enzymes have evolved several mechanisms to cleave peptide bonds. These distinct types have been systematically categorized in the MEROPS database. While a BLAST search on these proteases identifies homologous proteins, sequence alignment methods often fail to identify relationships arising from convergent evolution, exon shuffling, and modular reuse of catalytic units. We have previously established a computational method to detect functions in proteins based on the spatial and electrostatic properties of the catalytic residues (CLASP). CLASP identified a promiscuous serine protease scaffold in alkaline phosphatases (AP) and a scaffold recognizing a β-lactam (imipenem) in a cold-active Vibrio AP. Subsequently, we defined a methodology to quantify promiscuous activities in a wide range of proteins. Here, we assemble a module which encapsulates the multifarious motifs used by protease families listed in the MEROPS database. Since APs and proteases are an integral component of outer membrane vesicles (OMV), we sought to query other OMV proteins, like phospholipase C (PLC), using this search module. Our analysis indicated that phosphoinositide-specific PLC from Bacillus cereus is a serine protease. This was validated by protease assays, mass spectrometry and by inhibition of the native phospholipase activity of PI-PLC by the well-known serine protease inhibitor AEBSF (IC50 = 0.018 mM). Edman degradation analysis linked the specificity of the protease activity to a proline in the amino terminal, suggesting that the PI-PLC is a prolyl peptidase. Thus, we propose a computational method of extending protein families based on the spatial and electrostatic congruence of active site residues.

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., (© 2024 International Union of Biochemistry and Molecular Biology, Inc.)

A serine protease called prolyl endopeptidase (PEP) hydrolyses the peptide bonds on the carboxy side of the proline ring. The excessive PEP expression in brain results in neurodegenerative illnesses like dementia, Alzheimer's disease, and Parkinson's disease. Results of the prior studies on antioxidant activity, and the non-cytotoxic effect of bi-carbazole-linked triazoles, encouraged us to extend our studies towards its anti-diabetic potential. Hence, for this purpose all compounds 1-9 were evaluated to reveal their anti-prolyl endo peptidase activity. Fortunately, seven compounds resulted into significant inhibitory capability ranging from 26 to 63 µM. Among them six compounds 4-9 exhibited more potent inhibitory activity with IC 50 values 46.10 ± 1.16, 42.30 ± 1.18, 37.14 ± 1.21, 26.29 ± 0.76, 28.31 ± 0.64 and 31.11 ± 0.84 µM respectively, while compound 3 was the least active compound in the series with IC 50 value 63.10 ± 1.58 µM comparing with standard PEP inhibitor bacitracin (IC 50  = 125 ± 1.50 µM). Moreover, mechanistic study was performed for the most active compounds 7 and 8 with K i values 24.10 ± 0.0076 and 23.67 ± 0.0084 µM respectively. Further, the in silico studies suggested that the compounds exhibited potential interactions and significant molecular conformations, thereby elucidating the structural basis for their inhibitory effects., (© 2024. The Author(s).)

BackgroundMaternal genotype has lifetime effects on progeny, but few specific genes, and no proteases, are known to underlie maternal effects. Prolyl endopeptidase (PREP) is a serine protease with putative substrates that regulate appetite or milk production.ObjectiveTo test effects of PREP on obesity phenotypes in mice.DesignMice with a gene trap (GT) of PREP (PREP(gt/gt)) on the C57BL/6J (B6) background were generated. Minimal PREP protein was detected by western blot. In Experiment 1, direct effects of PREP were measured in littermate mice derived from intercrosses of heterozygotes (PREP(WT/gt)). In Experiment 2, maternal effects of PREP were measured in reciprocal crosses of heterozygous (PREP(WT/gt)) and wild-type (WT) (PREP(WT/WT)) males and females. DIETS: Mice were fed either low-fat (LF, Experiments 1 and 2) or high-fat (HF, Experiment 1) defined diets.MeasurementsAdiposity index (AI) was calculated from body weight (BW) and weights of four fat depots measured in 120-day-old mice. Fasting plasma glucose, insulin and leptin were measured. In vivo plasma alpha-MSH levels were measured by targeted quantitative peptidomics.ResultsExperiment 1-In intercross mice, there were significant diet effects, but few genotype effects. There were no genotype effects on BW or AI in males or females on either diet. Experiment 2-In contrast, reciprocal crosses of heterozygous males or females with WT B6 revealed highly significant parent of origin effects on all traits except body length. Progeny (WT and heterozygous genotypes and both sexes) born to female PREP(WT/gt) heterozygotes had fat pads that weighed as much as -twofold more at 120 days old than progeny born to male heterozygotes.ConclusionHeterozygosity for PREP GT results in highly significant maternal effects, whereas homozygosity for the PREP(gt/gt) mutation has a much more limited direct effect.

BackgroundIn vitro reactions are useful to identify putative enzyme substrates, but in vivo validation is required to identify actual enzyme substrates that have biological meaning. To investigate in vivo effects of prolyl endopeptidase (PREP), a serine protease, on alpha melanocyte stimulating hormone (alpha-MSH), we developed a new mass spectrometry based technique to quantitate, in multiplex, the various forms of alpha-MSH.MethodsUsing Multiple Reaction Monitoring (MRM), we analyzed peptide transitions to quantify three different forms of alpha-MSH. Transitions were first confirmed using standard peptides. Samples were then analyzed by mass spectrometry using a triple quadrupole mass spectrometer, after elution from a reverse phase C18 column by a gradient of acetonitrile.ResultsWe first demonstrate in vitro that PREP digests biological active alpha melanocyte stimulating hormone (alpha-MSH(1-13)), by cleaving the terminal amidated valine and releasing a truncated alpha melanocyte stimulating hormone (alpha-MSH(1-12)) product--the 12 residues alpha-MSH form. We then use the technique in vivo to analyze the MRM transitions of the three different forms of alpha-MSH: the deacetylated alpha-MSH(1-13), the acetylated alpha-MSH(1-13) and the truncated form alpha-MSH(1-12). For this experiment, we used a mouse model (PREP-GT) in which the serine protease, prolyl endopeptidase, is deficient due to a genetrap insertion. Here we report that the ratio between acetylated alpha-MSH(1-13) and alpha-MSH(1-12) is significantly increased (P-value = 0.015, N = 6) in the pituitaries of PREP-GT mice when compared to wild type littermates. In addition no significant changes were revealed in the relative level of alpha-MSH(1-13) versus the deacetylated alpha-MSH(1-13). These results combined with the demonstration that PREP digests alpha-MSH(1-13) in vitro, strongly suggest that alpha-MSH(1-13) is an in vivo substrate of PREP.ConclusionThe multiplex targeted quantitative peptidomics technique we present in this study will be decidedly useful to monitor several neuropeptide enzymatic reactions in vivo under varying conditions.

Proline-specific endoproteases have been successfully used in, for example, the in-situ degradation of gluten, the hydrolysis of bitter peptides, the reduction of haze during beer production, and the generation of peptides for mass spectroscopy and proteomics applications. Here we present the crystal structure of the extracellular proline-specific endoprotease from Aspergillus niger (AnPEP), a member of the S28 peptidase family with rarely observed true proline-specific endoprotease activity. Family S28 proteases have a conventional Ser-Asp-His catalytic triad, but their oxyanion-stabilizing hole shows a glutamic acid, an amino acid not previously observed in this role. Since these enzymes have an acidic pH optimum, the presence of a glutamic acid in the oxyanion hole may confine their activity to an acidic pH. Yet, considering the presence of the conventional catalytic triad, it is remarkable that the A. niger enzyme remains active down to pH 1.5. The determination of the primary cleavage site of cytochrome c along with molecular dynamics-assisted docking studies indicate that the active site pocket of AnPEP can accommodate a reverse turn of approximately 12 amino acids with proline at the S1 specificity pocket. Comparison with the structures of two S28-proline-specific exopeptidases reveals not only a more spacious active site cavity but also the absence of any putative binding sites for amino- and carboxyl-terminal residues as observed in the exopeptidases, explaining AnPEP's observed endoprotease activity., (© 2023 The Authors. Protein Science published by Wiley Periodicals LLC on behalf of The Protein Society.)

Prolyl oligopeptidase (POP) is a conserved serine protease belonging to proline-specific peptidases. It has both enzymatic and non-enzymatic activity and is involved in numerous biological processes in the human body, playing a role in e.g., cellular growth and differentiation, inflammation, as well as the development of some neurodegenerative and neuropsychiatric disorders. This article describes the physiological and pathological aspects of POP activity and the state-of-art of its peptidic inhibitors originating from food proteins, with a particular focus on their potential as cognition-enhancing agents. Although some milk, meat, fish, and plant protein-derived peptides have the potential to be applied as natural, procognitive nutraceuticals, their effectiveness requires further evaluation, especially in clinical trials. We demonstrated that the important features of the most promising POP-inhibiting peptides are very short sequence, high content of hydrophobic amino acids, and usually the presence of proline residue.

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.

Ethnopharmacological Relevance: Cotinus coggygria has a number of applications in traditional medicine most of which are connected with its anti-inflammatory and anti-oxidant properties. Since inflammation and oxidative stress are recognized as triggering factors for cancer, anti-cancer activity has also been documented and the possible mechanisms of this activity are under investigation. Important components of C. coggygria extracts are shown to be hydrolysable gallotannins of which pentagalloyl-O-glucose has been studied in details. This compound inhibits various enzymes including prolyl oligopeptidase which is involved in tumorigenesis and tumour growth. According to our pilot studies, oligo-O-galloylglucoses with more than five galloyl residues are also presented in the herb of Bulgarian origin, but their activities have not been examined., Aim of the Study: To establish an extraction method by which it is possible to concentrate high molecular hydrolysable gallotannins from dried leaves of Cotinus coggygria and to determine their inhibitory properties towards prolyl oligopeptidase and fibroblast activation protein α., Materials and Methods: Dried leaves of C. coggygria were extracted using different solvents in single-phase or biphasic systems under various extraction conditions. Main compounds of the extracts were identified by using high performance liquid chromatography and liquid chromatography - high resolution mass spectrometry. The extracts' inhibitory properties towards prolyl oligopeptidase and fibroblast activation protein α were studied on recombinant human enzymes by enzyme kinetic analyses using a fluorogenic substrate., Results: Ethyl acetate/water (pH 3.0) extraction of dried plant leaves proved to be the most efficient method for isolation of high molecular hydrolysable gallotannins which can be further concentrated by precipitation of dicyclohexylammonium salts in ethyl acetate. The main components of those extracts were oligo-O-galloyl glucoses with more than five gallic acid residues. They were shown to inhibit both enzymes studied but were about 30 times more effective inhibitors of prolyl oligopeptidase., Conclusions: C. coggygria from Bulgarian origin is shown to possess a substantial quantity of oligo-O-galloyl glucoses with more than five gallic acid residues which has not been described thus far in the same herb from other sources. An extraction method useable for concentrating those compounds is established. They are found to inhibit prolyl oligopeptidase with a very good selectivity to fibroblast activation protein α. The previously described antitumor activity of this plant may be at least in part due to the inhibition of the above enzymes which has been shown to participate in the genesis and development of various types of tumors., Competing Interests: Declaration of competing interest The authors declare no conflicts of interest., (Copyright © 2023 The Authors. Published by Elsevier B.V. All rights reserved.)

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.

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., (© 2023. BioMed Central Ltd., part of Springer Nature.)

Macrophages are immune cells crucial for host defense and homeostasis maintenance, and their dysregulation is involved in multiple pathological conditions, such as liver fibrosis. The transcriptional regulation in macrophage is indispensable for fine-tuning of macrophage functions, but the details have not been fully elucidated. Prolyl endopeptidase (PREP) is a dipeptidyl peptidase with both proteolytic and non-proteolytic functions. In this study, we found that Prep knockout significantly contributed to transcriptomic alterations in quiescent and M1/M2-polarized bone marrow-derived macrophages (BMDMs), as well as aggravated fibrosis in an experimental nonalcoholic steatohepatitis (NASH) model. Mechanistically, PREP predominantly localized to the macrophage nuclei and functioned as a transcriptional coregulator. Using CUT&Tag and co-immunoprecipitation, we found that PREP was mainly distributed in active cis-regulatory genomic regions and physically interacted with the transcription factor PU.1. Among PREP-regulated downstream genes, genes encoding profibrotic cathepsin B and D were overexpressed in BMDMs and fibrotic liver tissue. Our results indicate that PREP in macrophages functions as a transcriptional coregulator that finely tunes macrophage functions, and plays a protective role against liver fibrosis pathogenesis., (© 2023. The Author(s).)

Prolyl oligopeptidase (PREP) is a widely distributed serine protease in the human body cleaving proline-containing peptides; however, recent studies suggest that its effects on pathogenic processes underlying neurodegeneration are derived from direct protein-protein interactions (PPIs) and not from its regulation of certain neuropeptide levels. We discovered novel nonpeptidic oxazole-based PREP inhibitors, which deviate from the known structure-activity relationship for PREP inhibitors. These new compounds are effective modulators of the PPIs of PREP, reducing α-synuclein (αSyn) dimerization and enhancing protein phosphatase 2A activity in a concentration-response manner, as well as reducing reactive oxygen species production. From the best performing oxazoles, HUP-55 was selected for in vivo studies. Its brain penetration was evaluated, and it was tested in αSyn virus vector-based and αSyn transgenic mouse models of Parkinson's disease, where it restored motor impairment and reduced levels of oligomerized αSyn in the striatum and substantia nigra .

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., (© 2023 Wiley-VCH GmbH.)

Tauopathies are neurodegenerative diseases that are characterized by accumulation of hyperphosphorylated tau protein, higher-order aggregates, and tau filaments. Protein phosphatase 2A (PP2A) is a major tau dephosphorylating phosphatase, and a decrease in its activity has been demonstrated in tauopathies, including Alzheimer's disease. Prolyl oligopeptidase is a serine protease that is associated with neurodegeneration, and its inhibition normalizes PP2A activity without toxicity under pathological conditions. Here, we assessed whether prolyl oligopeptidase inhibition could protect against tau-mediated toxicity in cellular models in vitro and in the PS19 transgenic mouse model of tauopathy carrying the human tau-P301S mutation. We show that inhibition of prolyl oligopeptidase with the inhibitor KYP-2047 reduced tau aggregation in tau-transfected HEK-293 cells and N2A cells as well as in human iPSC-derived neurons carrying either the P301L or tau-A152T mutation. Treatment with KYP-2047 resulted in increased PP2A activity and activation of autophagic flux in HEK-293 cells and N2A cells and in patient-derived iNeurons, as indicated by changes in autophagosome and autophagy receptor markers; this contributed to clearance of insoluble tau. Furthermore, treatment of PS19 transgenic mice for 1 month with KYP-2047 reduced tau burden in the brain and cerebrospinal fluid and slowed cognitive decline according to several behavioral tests. In addition, a reduction in an oxidative stress marker was seen in mouse brains after KYP-2047 treatment. This study suggests that inhibition of prolyl oligopeptidase could help to ameliorate tau-dependent neurodegeneration.

Prolyl oligopeptidases (POPs) are serine proteases found in prokaryotes and eukaryotes which hydrolyze the peptide bond containing proline. The current study focuses on the analysis of POP sequences, their distribution and domain architecture in Shewanella woodyi, a Gram-negative, luminous bacterium which causes celiac sprue and similar infections in marine organisms. The POP undergoes huge interdomain movement, which allows possible route for the entry of any substrate. Hence, it offers an opportunity to understand the mechanism of substrate gating by studying the domain architecture and possibility to identify a probable drug target. In the present study, the POP sequence was retrieved from GenBank database and the best homologous templates were identified by PSI-BLAST search. The three-dimensional structures of the closed and open forms of POP from S. woodyi, which are not available in native form, were generated by homology modeling. The ideal lead molecules were screened by computer-aided virtual screening, and the binding potential of the best leads toward the target was studied by molecular docking. The domain architecture of the POP revealed that it has a propeller domain consists of $$\beta$$ -sheets, surrounded by $$\alpha$$ -helices and $$\alpha /\beta$$ hydrolase domain with catalytic triad containing Ser-564, Asp-646 and His-681. The hypothetical models of open and closed POP showed backbone RMSD value of 0.56 and 0.65 Å, respectively. Ramachandran plot of the open and closed POP conformations accounts for 99.4 and 98.7 % residues in the favoured region, respectively. Our study revealed that propeller domain comes as an insert between N-terminal and C-terminal $$\alpha /\beta$$ hydrolase domain. Molecular docking, drug likeness properties and ADME prediction suggested that KUC-103481N and Pramiracetum can be used as probable lead molecules toward the POP from S. woodyi. [ABSTRACT FROM AUTHOR]

Prolyl oligopeptidase (POP) plays a crucial role in the processing and degradation of neuropeptides and regulates inositol trisphosphate (IP3) signaling in mammals. We have reported that POP inhibition leads to IP3-mediated calcium efflux leading to mitochondrial-mediated apoptosis in the filarial parasite Setaria cervi. This study further elucidates the effect of altered calcium homeostasis on the proteome of filarial parasites. Adult parasites were treated with POP's specific inhibitor, Z-Pro-prolinal (ZPP), for 7 h. Cytosolic and mitochondrial proteome was analyzed using 2D gel electrophoresis coupled with MALDI-MS/MS. Phosphoproteins were also analyzed in the cytosolic fraction of the parasites. The phosphoprotein analysis revealed 7, and 9 spots in the cytosolic fraction of control and ZPP-treated parasites, respectively. The two identified protein spots in the treated set were found to be involved in G protein signaling. In cytosolic fraction, 109 and 112 protein spots were observed in control and treated parasites, respectively. Of these, 56 upregulated and 32 downregulated protein spots were observed in the treated set. On the other hand, 50 and 47 protein spots were detected in the mitochondrial fraction of control and treated parasites, respectively. Of these spots, 18 upregulated and 12 down-regulated protein spots were found in treated parasites. In silico analysis showed that the identified proteins were involved in energy metabolism, calcium signaling, stress response, and cytoskeleton organization. These findings correlate with our previous results suggesting the important regulatory role of POP in signaling and different metabolic pathways of filarial parasites., (© 2022. The Author(s), under exclusive licence to Springer Science+Business Media, LLC, part of Springer Nature.)

The objective of this study was to determine the in vitro activities such as antioxidant and inhibitions of angiotensin converting enzyme, dipeptidyl peptidase-IV, prolyl oligopeptidase, and 3-hydroxy-3-methyl-glutaryl-coenzyme A reductase of sacha inchi protein hydrolysate (SPH) and its membrane ultrafiltration peptide fractions. SPH was prepared after hydrolysis of sacha inchi protein using papain followed by separation into peptide fractions (F1: <1 kDa, F2: 1-3 kDa, F3: 3-5 kDa, and F4: 5-10 kDa) via ultrafiltration membranes. SPH and the peptide fractions were tested for multifunctional properties, specifically functional ability as antioxidants and enzyme inhibitors. Surface hydrophobicity was an important contributing factor to the activity of antioxidative peptides. The DPPH inhibitory activity of F4 was significantly higher (p < .05) than activities of the SPH and other fractions. The smaller peptides with <1 kDa size (F1) showed the most potent (p < .05) antioxidant properties based on the stronger scavenging of ABTS, DPPH, and superoxide radicals in addition to better attenuation of linoleic acid peroxidation. Moreover, the F1 was also the strongest inhibitor of angiotensin converting enzyme, dipeptidyl peptidase-IV, prolyl oligopeptidase inhibition, and 3-hydroxy-3-methyl-glutaryl-coenzyme A reductase based on the lower IC 50 values. It was concluded that the smaller size of the F1 peptides was the main determinant of its strong antioxidant and enzyme inhibition potency, which could be taken as an advantage to formulate functional foods and nutraceuticals with potential activities in ameliorating some of the chronic human diseases. PRACTICAL APPLICATIONS: The results of present study indicate that SPH and its ultrafiltration fractions are potential sources of antihypertensive, antidiabetic, inhibition of POP, reduced cholesterol, and strong antioxidant peptides. The strong angiotensin converting enzyme, dipeptidyl peptidase-IV, prolyl oligopeptidase inhibition, and 3-hydroxy-3-methyl-glutaryl-coenzyme inhibitory efficiency of the F1 peptides (MW < 1 kDa) suggest potential utility as an antihypertensive, antidiabetic agent, reduce cholesterol and brain plasticity and memory formation because the small peptide size could enhance absorption from the gastrointestinal tract. Overall, results from this study indicate that SPH, especially the F1 peptides may have applications as ingredients for the formulation of functional foods and nutraceuticals., (© 2022 Wiley Periodicals LLC.)

Parkinson's disease (PD) is characterized by degeneration of nigrostriatal dopaminergic neurons and accumulation of α-synuclein (αSyn) as Lewy bodies. Currently, there is no disease-modifying therapy available for PD. We have shown that a small molecular inhibitor for prolyl oligopeptidase (PREP), KYP-2047, relieves αSyn-induced toxicity in various PD models by inducing autophagy and preventing αSyn aggregation. In this study, we wanted to study the effects of PREP inhibition on different αSyn species by using cell culture and in vivo models. We used Neuro2A cells with transient αSyn overexpression and oxidative stress or proteasomal inhibition-induced αSyn aggregation to assess the effect of KYP-2047 on soluble αSyn oligomers and on cell viability. Here, the levels of soluble αSyn were measured by using ELISA, and the impact of KYP-2047 was compared to anle138b, nilotinib and deferiprone. To evaluate the effect of KYP-2047 on αSyn fibrillization in vivo, we used unilateral nigral AAV1/2-A53T-αSyn mouse model, where the KYP-2047 treatment was initiated two- or four-weeks post injection. KYP-2047 and anle138b protected cells from αSyn toxicity but interestingly, KYP-2047 did not reduce soluble αSyn oligomers. In AAV-A53T-αSyn mouse model, KYP-2047 reduced significantly proteinase K-resistant αSyn oligomers and oxidative damage related to αSyn aggregation. However, the KYP-2047 treatment that was initiated at the time of symptom onset, failed to protect the nigrostriatal dopaminergic neurons. Our results emphasize the importance of whole αSyn aggregation process in the pathology of PD and raise an important question about the forms of αSyn that are reasonable targets for PD drug therapy., Competing Interests: Declaration of competing interest The authors have no competing interests to declare that are relevant to the content of this article., (Copyright © 2022 The Authors. Published by Elsevier Ltd.. All rights reserved.)

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., (© 2022. The Author(s).)

We have previously described several different chemical series of bicyclic prolyl oligopeptidase (POP) inhibitors as probes for neurodegenerative diseases that demonstrated nanomolar activity in vitro and submicromolar activity in cellulo. The more recent implication of POP in cancer, together with homologous fibroblast activation protein α (FAP), implicated in tumor growth, led us to consider developing POP/FAP dual inhibitors as a promising strategy for the development of cancer therapeutics. At this stage, we thought to evaluate the requirements for selectivity of inhibitors for POP over FAP and to evaluate molecular platforms that would enable the development of selective POP and dual POP/FAP inhibitors. We report herein docking-guided design of a new bicyclic scaffold and synthesis of both covalent and non-covalent bicyclic inhibitors. Biological evaluation of first-of-their-kind [4.3.0] bicyclic compounds confirmed that reactive groups, or covalent warheads, are required for inhibitor activity. This work ultimately led to one scaffold yielding new POP-selective inhibitors and a dual inhibitor equipotent to the only drug targeting FAP and POP that ever reached clinical trials., (Copyright © 2022 Elsevier Masson SAS. All rights reserved.)

Background: A gluten-free diet (GFD) is the main therapy for non-coeliac gluten sensitivity (NCGS). However, the availability of novel enzymes with the ability to digest gluten could represent a therapeutic opportunity for NCGS patients to avoid a GFD., Aims: To evaluate the controlled reintroduction of gluten with or without the endopeptidase P1016 in NCGS patients., Methods: This is a randomized, double-blind, placebo-controlled monocentric study, Registered under ClinicalTrials.gov Identifier no. NCT01864993. Gluten was reintroduced incrementally over a 3-week period under nutritional control. NCGS patients were randomized into two groups and administered P1016 or placebo during gluten reintroduction. We evaluated symptoms (visual analogue scale, VAS), quality of life (SF-36) and mental health symptoms (SCL-90) on a weekly basis., Results: We enrolled a total 23 patients who were allocated to a placebo group (n = 11, age 38.4 ± 2.9) or an intervention group (n = 12, age 39.5 ± 3.1). No effect of P1016 on symptoms was found. During gluten reintroduction, patients reported a significant increase in abdominal pain and a worsening of stool consistency. Furthermore, no differences were found between the groups regarding SCL-90 and SF-36 scores., Conclusions: Our results demonstrate a lack of effect of P1016 in the management of NCGS patients and the possible reintroduction of gluten., Competing Interests: Conflict of interest The authors declare no conflict of interest., (Copyright © 2022 Elsevier Ltd and European Society for Clinical Nutrition and Metabolism. All rights reserved.)

The digestion of gluten generates toxic peptides, among which a highly immunogenic proline-rich 33-mer from wheat α-gliadin, that trigger coeliac disease. Neprosin from the pitcher plant is a reported prolyl endopeptidase. Here, we produce recombinant neprosin and its mutants, and find that full-length neprosin is a zymogen, which is self-activated at gastric pH by the release of an all-β pro-domain via a pH-switch mechanism featuring a lysine plug. The catalytic domain is an atypical 7+8-stranded β-sandwich with an extended active-site cleft containing an unprecedented pair of catalytic glutamates. Neprosin efficiently degrades both gliadin and the 33-mer in vitro under gastric conditions and is reversibly inactivated at pH > 5. Moreover, co-administration of gliadin and the neprosin zymogen at the ratio 500:1 reduces the abundance of the 33-mer in the small intestine of mice by up to 90%. Neprosin therefore founds a family of eukaryotic glutamate endopeptidases that fulfils requisites for a therapeutic glutenase., (© 2022. The Author(s).)

Prolyl endopeptidase or prolyl oligopeptidase (PEP or POP) is highly expressed in brain, and associated with autism spectrum disorders, dementia, aging and various psychological disorders, such as schizophrenia, mania, and neurodegeneration. To design highly potent and novel POP inhibitors, structure-based virtual screening was carried out using pharmacophore modeling and molecular docking studies. The docking based active compounds [incensole (1), incensole acetate (2), incensone (3), incensfuran (4), and epi-incensole acetate (5)] were selected and their dynamic behavior was studied through molecular dynamic simulation. Later, the top-ranked [predicted active, (1-5)] and lower-ranked [predicted in-active, (6-10)] compounds were tested by in-vitro assay. The in-vitro results showed that all top-ranked compounds (1-5) found significantly active against POP enzyme with IC 50 values in range of 3.1 ± 0.45 to 24.4 ± 1.16 μM, while lower-ranked (6-10) were inactive, indicated accuracy of docking results. Kinetics studies on all active compounds 1-5 were carried out to investigate their mode of inhibition and dissociation constants K i . All compounds showed competitive behaviors with K i values in the range of 0.92-8.12 μM. The study resulted in the identification of five (1-5) diterpene based molecules from natural sources that significantly inhibit the activity of POP by competitive mode of inhibition., (Copyright © 2022. Published by Elsevier B.V.)

Soy sauce secondary precipitate formed due to the B 3 subunit seriously affects soy sauce's appearance quality. In this study, a prolyl endopeptidase (APE) from Aspergillus niger , which could degrade approximately 50% of the B 3 subunit and increase proline content by 24% in soy sauce, was isolated and identified. The results showed that APE was an acidic salt-tolerant serine protease (62 kDa), which was optimally active at 40 °C and pH 4.0, and retained more than 69% activity in 3 M NaCl solution over 10 days. As a potential substrate of APE, the B 3 subunit contains 10 proline residues. High salinity could not damage the hydrogen bonds, salt bridges, and interior hydrophobicity of APE; thus, the spatial structures and activity of APE in 3 M NaCl solution were stable within 3 days and decreased thereafter. High salinity made the B 3 subunit more rigid and lowered the catalytic activity of APE on the B 3 subunit, hindering complete hydrolysis of the B 3 subunit. This was the first report about the APE capable of degrading the B 3 subunit and reducing the secondary precipitate of soy sauce, providing a new possibility to solve the secondary precipitate of soy sauce.

A healthy microenvironment of the intervertebral disc tissue is characterized by hypoxia owing to its sparse vascular distribution. Oxidative stress plays a pivotal role in the pathological development of intervertebral disc degeneration (IVDD). We found that the expression of prolyl endopeptidase (PREP) increased in degenerative nucleus pulposus (NP) tissues. The purpose of this study was to determine whether PREP is involved in oxidative-stress-induced IVDD. Tertbutyl hydroperoxide can inhibit the expression of PREP by activating the PI3K/AKT signaling pathway at low concentrations in NP cells. Knockdown of PREP protected NP cells from apoptosis induced by oxidative stress, whereas overexpression of PREP exacerbated the apoptosis of NP cells. We also investigated the connection between the PI3K/AKT signaling pathway and PREP and found that the activation of the PI3K/AKT signaling pathway downregulated the expression of PREP by inhibiting p53. As a crucial transcription factor, p53 binds to the PREP promoter region and promotes its transcription. Overexpression of PREP also impairs protein secretion in the extracellular matrix of NP cells. Furthermore, the in vivo knockout of PREP could attenuate puncture-induced IVDD. These findings suggested that the downregulation of PREP might maintain the viability of NP cells and attenuate IVDD under oxidative stress., Competing Interests: The authors confirm no conflicts of interest., (Copyright © 2022 Huo-Liang Zheng et al.)

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.

There is increasing interest in gluten-degrading enzymes for use during food and drink processing. The industrially available enzymes usually work best at low to ambient temperatures. However, food manufacturing is often conducted at higher temperatures. Therefore, thermostable gluten-degrading enzymes are of great interest. We have identified a new thermostable gluten-degrading proline-specific prolyl endoprotease from the archaea Thermococcus kodakarensis. We then cloned and expressed it in Escherichia coli. The prolyl endoprotease was found to have a size of 70.1 kDa. The synthetic dipeptide Z-Gly-Pro-p-nitroanilide was used to characterize the prolyl endoprotease and it had maximum activity at pH 7 and 77°C. The Vmax, Km and kcat values of the purified prolyl endoprotease were calculated to be 3.14 mM/s, 1.10 mM and 54 s-1, respectively. When the immunogenic gluten peptides PQPQLPYPQPQLPY (α-gliadin) and SQQQFPQPQQPFPQQP (γ-hordein) were used as substrates, the prolyl endoprotease was able to degrade these. Furthermore, gluten in wort was reduced when the prolyl endoprotease was used during mashing of barley malt. The discoveries open up new food processing possibilities and further the understanding of proline-specific protease diversity., (© The Author(s) 2022. Published by Oxford University Press on behalf of FEMS.)

Oxidative stress (OS) is a common toxic feature in various neurodegenerative diseases. Therefore, reducing OS could provide a potential approach to achieve neuroprotection. Prolyl oligopeptidase (PREP) is a serine protease that is linked to neurodegeneration, as endogenous PREP inhibits autophagy and induces the accumulation of detrimental protein aggregates. As such, inhibition of PREP by a small-molecular inhibitor has provided neuroprotection in preclinical models of neurodegenerative diseases. In addition, PREP inhibition has been shown to reduce production of reactive oxygen species (ROS) and the absence of PREP blocks stress-induced ROS production. However, the mechanism behind PREP-related ROS regulation is not known. As we recently discovered PREP's physiological role as a protein phosphatase 2A (PP2A) regulator, we wanted to characterize PREP inhibition as an approach to reduce OS. We studied the impact of a PREP inhibitor, KYP-2047, on hydrogen peroxide and ferrous chloride induced ROS production and on cellular antioxidant response in HEK-293 and SH-SY5Y cells. In addition, we used HEK-293 and SH-SY5Y PREP knock-out cells to validate the role of PREP on stress-induced ROS production. We were able to show that absence of PREP almost entirely blocks the stress-induced ROS production in both cell lines. Reduced ROS production and smaller antioxidant response was also seen in both cell lines after PREP inhibition by 10 μM KYP-2047. Our results also revealed that the OS reducing mechanism of PREP inhibition is related to reduced activation of ROS producing NADPH oxidase through enhanced PP2A activation. In conclusion, our results suggest that PREP inhibition could also provide neuroprotection by reducing OS, thus broadening the scope of its beneficial effects on neurodegeneration., (Copyright © 2021 The Author(s). Published by Elsevier Inc. All rights reserved.)

Aimed to study the characteristics of prolyl endopeptidase (PEP, EC 3.4.21.26) and its possible role in the degradation of collagen, we cloned the full-length cDNA sequence of PEP from abalone (Haliotis discus hannai) (Hdh-PEP). Recombinant Hdh-PEP (rHdh-PEP) was expressed in vitro, its enzymatic properties were detected, and its secondary structure was analyzed by Circular Dichroism (CD). We for the first time determined the 1.5 Å crystal structure of rHdh-PEP. The decomposition effect of rHdh-PEP on collagen peptides was analyzed. Our data revealed that the molecular weight of rHdh-PEP is 85 kDa, consisting of a catalytic domain and a β-propeller domain. The optimal pH and temperature of rHdh-PEP were pH 6.0 and 20 °C, respectively. Using small collagen peptides as substrates, HPLC-ESI-MS analysis confirmed that rHdh-PEP specifically cleaved at the carboxyl side of proline residues, suggesting its role in the degradation of collagen peptides during autolysis., Competing Interests: Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2020 Elsevier Ltd. All rights reserved.)

A selective prolyl endopeptidase (PEP) inhibitor benzyloxycarbonyl-prolyl-prolinal (IC50 = 1,61±0,12 nmol/l) and a nonselective PEP inhibitor benzyloxycarbonyl-methionyl-cyanopyrrolidine (IC50 = 2,01±0,14 nmol/l) exhibit a comparable antiexudative effect at single doses of 2 mg/kg and 5 mg/kg (intraperitoneally) in outbred mice with peritonitis induced by 1% acetic acid. However, only benzyloxycarbonyl-methionyl-cyanopyrrolidine at a dose of 5 mg/kg reduces acetic acid induced pain in animals.

Fibroblast activation protein (FAP) is a non-classical serine protease expressed predominantly in conditions accompanied by tissue remodeling, particularly cancer. Due to its plasma membrane localization, FAP represents a promising molecular target for tumor imaging and treatment. The unique enzymatic activity of FAP facilitates development of diagnostic and therapeutic tools based on molecular recognition of FAP by substrates and small-molecule inhibitors, in addition to conventional antibody-based strategies. In this review, we provide background on the pathophysiological role of FAP and discuss its potential for diagnostic and therapeutic applications. Furthermore, we present a detailed analysis of the structural patterns crucial for substrate and inhibitor recognition by the FAP active site and determinants of selectivity over the related proteases dipeptidyl peptidase IV and prolyl endopeptidase. We also review published data on targeting of the tumor microenvironment with FAP antibodies, FAP-targeted prodrugs, activity-based probes and small-molecule inhibitors. We describe use of a recently developed, selective FAP inhibitor with low-nanomolar potency in inhibitor-based targeting strategies including synthetic antibody mimetics based on hydrophilic polymers and inhibitor conjugates for PET imaging. In conclusion, recent advances in understanding of the molecular structure and function of FAP have significantly contributed to the development of several tools with potential for translation into clinical practice., Competing Interests: Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2020. Published by Elsevier B.V.)

The proline-specific enzymes dipeptidyl peptidase 4 (DPP4), prolylcarboxypeptidase (PRCP), fibroblast activation protein α (FAP) and prolyl oligopeptidase (PREP) are known for their involvement in the immune system and blood pressure regulation. Only very limited information is currently available on their enzymatic activity and possible involvement in patients with sepsis and septic-shock. The activity of the enzymes was measured in EDTA-plasma of patients admitted to the intensive care unit (ICU): 40 septic shock patients (sepsis-2) and 22 ICU control patients after major intracranial surgery. These data were used to generate receiver operating characteristic (ROC) curves. A survival analysis (at 90 days) and an association study with other parameters was performed. PRCP (day 1) and PREP (all days) enzymatic activities were higher in septic shock patients compared to controls. In contrast, FAP and DPP4 were lower in these patients on all studied time points. Since large differences were found, ROC curves were generated and these yielded area under the curve (AUC) values for PREP, FAP and DPP4 of 0.88 (CI: 0.80-0.96), 0.94 (CI: 0.89-0.99) and 0.86 (CI: 0.77-0.95), respectively. PRCP had a lower predicting value with an AUC of 0.71 (CI: 0.58-0.83). A nominally significant association was observed between survival and the DPP4 enzymatic activity at day 1 (p<0.05), with a higher DPP4 activity being associated with an increase in survival. All four enzymes were dysregulated in septic shock patients. DPP4, FAP and PREP are good in discriminating between septic shock patients and ICU controls and should be further explored to see whether they are already dysregulated in earlier stages, opening perspectives for their further investigation as biomarkers in sepsis. DPP4 also shows potential as a prognostic biomarker. Additionally, the associations found warrant further research., Competing Interests: The authors have declared that no competing interests exist.

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)., Competing Interests: The authors have declared that no competing interests exist.

Prolyl oligopeptidase (POP) enzyme has been studied for various disorders, viz. Schizophrenia, Alzheimer's, Parkinson's, Depression, Inflammation, etc., for three decades, but no drug has passed through the clinical trials, possibly because of indigent pharmacokinetics. This might have been a result of similar structures of drug candidates. This study aimed at identifying novel small non-peptidomimetic inhibitors for POP enzyme that could serve as a lead for developing newer drugs. Structure-based virtual screening of molecules of MolMall database was conducted on the POP enzyme (PDB ID 3DDU) to identify potential hits. The hits identified were subjected to computational pharmacokinetic screening followed by molecular mechanics/generalized Born and surface area studies to estimate the binding free energy of the docked complexes. After that, nine hits were selected and tested for POP inhibitory activity, among which one compound MM 4 was found to be most potent with EC 50 of 100 µM. Compound MM 4 was further subjected to molecular dynamics simulations to study the overall stability of the ligand-protein complex. The compound interacted strongly with catalytic amino acid Arg643 by forming salt and water bridges; it also interacted well with amino acids Phe173, Arg252 and Met235. This study provides a lead molecule for further development of POP inhibitors.Communicated by Ramaswamy H. Sarma.

Amatoxins are ribosomally synthesized and post-translationally modified peptides (RiPPs) found in poisonous mushrooms. These cyclic peptides are potent inhibitors of RNA polymerase II transcriptional activity. Though the macrocyclization of amatoxin is extensively studied, little is known about its subsequent post-translational modifications. However, studies and the potential use of amatoxins has been deterred by the scarcity of the mushroom biomass. To overcome this issue, we sought to produce the α-amanitin in Escherichia coli. Genes encoding the amanitin precursor peptide (AMA1) and prolyl oligopeptidase (POPB) were separately cloned and expressed in E. coli. Fusion tags were attached to candidate proteins to improve expression and solubility. Purified AMA1 was processed in vitro by POPB, and the formation of cyclic α-amanitin was confirmed by HPLC and MALDI/TOF mass spectroscopy. Our strategy can be applied to the mass production of the α-amanitin, allowing α-amanitin to be investigated as a promising lead compound in drug development., (Copyright © 2019 Elsevier B.V. All rights reserved.)

The Ang II (Angiotensin II)-Angiotensin-(1-7) axis of the Renin Angiotensin System encompasses 3 enzymes that form Angiotensin-(1-7) [Ang-(1-7)] directly from Ang II: ACE2 (angiotensin-converting enzyme 2), PRCP (prolylcarboxypeptidase), and POP (prolyloligopeptidase). We investigated their relative contribution to Ang-(1-7) formation in vivo and also ex vivo in serum, lungs, and kidneys using models of genetic ablation coupled with pharmacological inhibitors. In wild-type (WT) mice, infusion of Ang II resulted in a rapid increase of plasma Ang-(1-7). In ACE2 -/- / PRCP -/- mice, Ang II infusion resulted in a similar increase in Ang-(1-7) as in WT (563±48 versus 537±70 fmol/mL, respectively), showing that the bulk of Ang-(1-7) formation in circulation is essentially independent of ACE2 and PRCP. By contrast, a POP inhibitor, Z-Pro-Prolinal reduced the rise in plasma Ang-(1-7) after infusing Ang II to control WT mice. In POP -/- mice, the increase in Ang-(1-7) was also blunted as compared with WT mice (309±46 and 472±28 fmol/mL, respectively P =0.01), and moreover, the rate of recovery from acute Ang II-induced hypertension was delayed ( P =0.016). In ex vivo studies, POP inhibition with ZZP reduced Ang-(1-7) formation from Ang II markedly in serum and in lung lysates. By contrast, in kidney lysates, the absence of ACE2, but not POP, obliterated Ang-(1-7) formation from added Ang II. We conclude that POP is the main enzyme responsible for Ang II conversion to Ang-(1-7) in the circulation and in the lungs, whereas Ang-(1-7) formation in the kidney is mainly ACE2-dependent.

Cyanopyrrolidine derivatives benzyloxycarbonyl-methionyl-cyanopyrrolidine (ZMetPrdN), benzyloxycarbonylphenylalanyl- cyanopyrrolidine (ZPhePrdN), tert-butyl-hydroxycarbonyl-glycyl-cyanopyrrolidine (BocGlyPrdN), tert-butyl-hydroxycarbonyl-methionyl-cyanopyrrolidine (BocMetPrdN) are inhibitors of prolylendopeptidase (PREP; EC 3.4.21.26) with an IC50 of 2 nM to 12 nM. ZMetPrdN, ZPhePrdN and BocMetPrdN additionally inhibited dipeptidyl peptidase IV (DPP-4; EC 3.4.14.5) with an IC50 of 1100 nM to 3200 nM. All the compounds have antinociceptive properties in the acetic acid writhing test in mice. But only cyanopyrrolidine derivatives with aromatic substituents decrease exudative inflammation. The cyanopyrrolidine derivatives also increase PREP activity and compensatorily reduce DPP-4 activity in the serum of mice three hours after the induction of inflammation. Thus, cyanopyrrolidine derivatives exhibit antinociceptive and antiexudative properties in part via their effect on PREP.

Over the past decade, many drug discovery endeavors have been invested in targeting the serine proteases prolyl oligopeptidase (POP) for the treatment of Alzheimer's and Parkinson's disease and, more recently, epithelial cancers. Our research group has focused on the discovery of reversible covalent inhibitors, namely nitriles, to target the catalytic serine residue in this enzyme. While there have been many inhibitors discovered containing a nitrile to covalently bind to the catalytic serine, we have been investigating others, particularly boronic acids and boronic esters, the latter of which have been largely unexplored as covalent warheads. Herein we report a series of computationally-designed POP boronic ester pro-drug inhibitors exhibiting nanomolar-potencies in vitro as their active boronic acid species. These easily-accessible (1-2 step syntheses) compounds could facilitate future biochemical and biological studies of this enzyme's role in neurodegenerative diseases and cancer progression., (Copyright © 2019 Elsevier Masson SAS. All rights reserved.)

D-Aspartate (D-Asp) is an endogenous amino acid that plays a central role in the development of the central nervous system (CNS) and functioning of the neuroendocrine system. In line with its functions, it is abundantly present in the CNS and reproductive systems of vertebrates and invertebrates. It has been implicated in the biosynthesis and/or secretion of hormones and factors that are involved in various reproductive functions, such as GnRH from the hypothalamus and testosterone from the testis. We conducted an in vivo study consisting of acute (i.p. injection of 2 µmol/g body weight) and chronic (15 days drinking solution) administration of D-Asp to adult rats to understand the signaling pathways elicited by D-Asp in the rat testis. We found that D-Asp upregulated the expression of prolyl endopeptidase (PREP), a serine protease having a pivotal role in the regulation of mammalian spermatogenesis and spermiogenesis. Immunofluorescence analysis revealed its overexpression in Leydig cells, Sertoli cells and spermatogonia. Moreover, PREP was found to co-localize with GluA2/3, an AMPA receptor subunit, whose protein expression also increased after D-Asp treatments. Finally, we found a significant increase in ERK and Akt activities in the testis of rats treated with D-Asp. Since PREP is known to be involved in regulating GnRH levels and in germ cell differentiation, we hypothesize D-Asp to play a pivotal role in regulating hormone homeostasis and spermatogenesis through activation of PREP, AMPAR, ERK and Akt.

Boc-Gly-Pro-DP, Z-Gly-Pro-DP, LA-Gly-Pro-DP, Boc-Gly-Pro-Srt, Z-Gly-Pro-Srt were synthesized for the first time. The stability of these compounds in the presence of leucine aminopeptidase, carboxypeptidase Y, carboxypeptidase B and proline endopeptidase (PEP) was determined. It turned out that the compounds are stable in the presence of aminopeptidases and carboxypeptidases. In the presence of PEP, dopamine (DP) and serotonin (Srt) are cleaved from the synthesized preparations. Thus, new proline-containing Srt and DP derivatives were obtained, Srt and DP could be gradually released from them. This suggest the possibility of a prolonged action of these biologically active compounds on the vital activity of cells and, consequently, of the whole organism.