Effect of Aspergillus niger prolyl endopeptidase in patients with celiac disease on a long-term gluten-free diet.

BACKGROUND: The gluten-free diet (GFD) has limitations, and there is intense research in the development of adjuvant therapies. AIM: To examine the effects of orally administered Aspergillus niger prolyl endopeptidase protease (AN-PEP) on inadvertent gluten exposure and symptom prevention in adult celiac disease (CeD) patients following their usual GFD. METHODS: This was an exploratory, double-blind, randomized, placebo-controlled trial that enrolled CeD patients on a long-term GFD. After a 4-wk run-in period, patients were randomized to 4 wk of two AN-PEP capsules (GliadinX; AVI Research, LLC, United States) at each of three meals per day or placebo. Outcome endpoints were: (1) Average weekly stool gluten immunogenic peptides (GIP) between the run-in and end of treatments and between AN-PEP and placebo; (2) celiac symptom index (CSI); (3) CeD-specific serology; and (4) quality of life. Stool samples were collected for GIP testing by ELISA every Tuesday and Friday during run-ins and treatments. RESULTS: Forty patients were randomized for the intention-to-treat analysis, and three were excluded from the per-protocol assessment. Overall, 628/640 (98.1%) stool samples were collected. GIP was undetectable (< 0.08 µg/g) in 65.6% of samples, and no differences between treatment arms were detected. Only 0.5% of samples had GIP concentrations sufficiently high (> 0.32 µg/g) to potentially cause mucosal damage. Median GIP concentration in the AN-PEP arm was 44.7% lower than in the run-in period. One-third of patients exhibiting GIP > 0.08 µg/g during run-in had lower or undetectable GIP after AN-PEP treatment. Compared with the run- in period, the proportion of symptomatic patients (CSI > 38) in the AN-PEP arm was significantly lower (P < 0.03). AN-PEP did not result in changes in specific serologies. CONCLUSION: This exploratory study conducted in a real-life setting revealed high adherence to the GFD. The AN-PEP treatment did not significantly reduce the overall GIP stool concentration. However, given the observation of a significantly lower prevalence of patients with severe symptoms in the AN-PEP arm, further clinical research is warranted.

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.

Evaluation of prolyl oligopeptidase and acetylcholinesterase inhibition by Phyllanthus tenellus Roxb. from Brazil.

Phyllanthus tenellus Roxb. (Phyllanthaceae) is a plant used in Brazilian folk medicine for the treatment of intestinal infections and diabetes. Despite its use in traditional medicine, it was reported that P. tenellus extract may cause several effects in the central nervous system (CNS) of animals, such as agitation and signs of depression. The aim of this study was to determine the main constituents of P. tenellus methanol extract and to investigate whether the extract is able to inhibit the enzymes prolyl oligopeptidase (POP), acetylcholinesterase (AChE) and dipeptidyl peptidase-IV (DPP-IV). Corilagin (1) was isolated as the main constituent of the P. tenellus extract, along with rutin (2) and vitexin-2″-O-rhamnoside (3). The extract presented the ability to inhibit mainly POP. Dichloromethane and ethyl acetate fractions showed the highest inhibitory potency against POP (IC50 values of 1.7 ± 0.4 and 11.7 ± 2 µg/mL, respectively). All fractions were inactive against AChE. Corilagin displayed selective POP inhibition in a dose-dependent manner, with IC50= 19.7 ± 2.6 µg/mL. Corilagin exhibited moderate capacity to pass through the phospholipid membrane by passive diffusion, presenting effective permeability (Pe) of 1.26 × 10-7 cm/s.

Prolyl Endopeptidase-Like Facilitates the α-Synuclein Aggregation Seeding, and This Effect Is Reverted by Serine Peptidase Inhibitor PMSF.

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

Chemical constituents from Valeriana polystachya Smith and evaluation of their effects on the acetylcholinesterase and prolyl oligopeptidase activities.

Two new iridoids (1-2) and a new decomposition product of valepotriates (3), together with fifteen known compounds (4-18) were isolated from the roots and rhizomes of Valeriana polystachya Smith, a native species from the Pampa Biome. Their structures were elucidated by means of NMR spectroscopy, mass spectrometry and optical rotation. The structures of 3 and 18 were further confirmed by single crystal X-ray diffraction analysis. In the group of the isolated compounds, 6ß-hydroxysitostenone, hydroxymaltol and isovillosol were isolated from the Valeriana genus for the first time. The extracts and isolated compounds were evaluated for their in vitro activities against acetylcholinesterase (AChE) and prolyloligopeptidase (POP). Compounds 7, 9 and 11 showed weak inhibitory activity against AChE, while 3 and 5 displayed exceptional POP inhibitory activity, with IC50 values of 5.3 ±â€¯0.07 and 7.9 ±â€¯0.4 µM, respectively.

Trypanosoma cruzi 80 kDa prolyl oligopeptidase (Tc80) as a novel immunogen for Chagas disease vaccine.

BACKGROUND: Chagas disease, also known as American Trypanosomiasis, is a chronic parasitic disease caused by the flagellated protozoan Trypanosoma cruzi that affects about 8 million people around the world where more than 25 million are at risk of contracting the infection. Despite of being endemic on 21 Latin-American countries, Chagas disease has become a global concern due to migratory movements. Unfortunately, available drugs for the treatment have several limitations and they are generally administered during the chronic phase of the infection, when its efficacy is considered controversial. Thus, prophylactic and/or therapeutic vaccines are emerging as interesting control alternatives. In this work, we proposed Trypanosoma cruzi 80 kDa prolyl oligopeptidase (Tc80) as a new antigen for vaccine development against Chagas disease. METHODOLOGY/PRINCIPAL FINDINGS: In a murine model, we analyzed the immune response triggered by different immunization protocols based on Tc80 and evaluated their ability to confer protection against a challenge with the parasite. Immunized mice developed Tc80-specific antibodies which were able to carry out different functions such as: enzymatic inhibition, neutralization of parasite infection and complement-mediated lysis of trypomastigotes. Furthermore, vaccinated mice elicited strong cell-mediated immunity. Spleen cells from immunized mice proliferated and secreted Th1 cytokines (IL-2, IFN-γ and TNF-α) upon re-stimulation with rTc80. Moreover, we found Tc80-specific polyfunctional CD4 T cells, and cytotoxic T lymphocyte activity against one Tc80 MHC-I peptide. Immunization protocols conferred protection against a T. cruzi lethal challenge. Immunized groups showed a decreased parasitemia and higher survival rate compared with non-immunized control mice. Moreover, during the chronic phase of the infection, immunized mice presented: lower levels of myopathy-linked enzymes, parasite burden, electrocardiographic disorders and inflammatory cells. CONCLUSIONS/SIGNIFICANCE: Considering that an early control of parasite burden and tissue damage might contribute to avoid the progression towards symptomatic forms of chronic Chagas disease, the efficacy of Tc80-based vaccines make this molecule a promising immunogen for a mono or multicomponent vaccine against T. cruzi infection.

The second point mutation in PREPL: a case report and literature review.

Prolyl endopeptidase-like (PREPL) deficiency (MIM# 616224) is a rare autosomal recessive inherited congenital myasthenic syndrome characterized by neonatal hypotonia, feeding problems, mild dysmorphism, and neuromuscular symptoms, followed by hyperphagia and obesity in later childhood. Some patients also exhibit growth deficits, sexual hormone deficiency, and cognitive impairments. This syndrome is caused by biallelic mutations in PREPL. To date, only one nucleotide deletion and seven small microdeletions in PREPL have been reported. Here we report a female patient with a novel homozygous frameshift mutation in PREPL (NM_006036.4, c.342delA:p.Val115Leufs*39). Her clinical features are similar to those of previously reported cases. The mutation is the first homozygous point mutation reported in humans.

Development of an Assay for Measuring Proprotein-Conversion Activity on a Multiplex Magnetic Bead-Based Array Platform.

This chapter describes a protocol for measuring prolyl oligopeptidase (POP) activity using a biotinylated peptide substrate coupled to magnetic microspheres. The complex is incubated in the presence of a pituitary extract and activity can be detected by loss of the biotin label. The assay can be multiplexed for measuring multiple proprotein-cleaving enzymes simultaneously and can be used in analyses of neuropsychiatric diseases in which proteolytic cleavage of biologically active peptides is known to play a role.

Chemical composition and evaluation of prolyl oligopeptidase and acetylcholinesterase inhibitory activities of Leonurus Sibiricus L. from Brazil.

Chemical investigation of the aerial parts of Leonurus sibiricus L. used in Brazilian folk medicine led to the identification of the following constituents: the labdane-type diterpenoid leojaponin, the phytosterols ß-sitosterol and ß-sitosterol glucoside and the alkaloid leonurine. The crude extracts obtained from methanol and methanol/1% HCl and pure compounds isolated from L. sibirius were investigated as acetylcholinesterase (AChE) and prolyl oligopeptidase (POP) inhibitors. Extracts obtained by maceration were active against POP (53-58%), but showed weak activity against AChE. The isolated leojaponin and leonurine were evaluated as POP inhibitors.

Identification of novel Trypanosoma cruzi prolyl oligopeptidase inhibitors by structure-based virtual screening.

We have previously demonstrated that the secreted prolyl oligopeptidase of Trypanosoma cruzi (POPTc80) is involved in the infection process by facilitating parasite migration through the extracellular matrix. We have built a 3D structural model where POPTc80 is formed by a catalytic α/ß-hydrolase domain and a ß-propeller domain, and in which the substrate docks at the inter-domain interface, suggesting a "jaw opening" gating access mechanism. This preliminary model was refined by molecular dynamics simulations and next used for a virtual screening campaign, whose predictions were tested by standard binding assays. This strategy was successful as all 13 tested molecules suggested from the in silico calculations were found out to be active POPTc80 inhibitors in the micromolar range (lowest K i at 667 nM). This work paves the way for future development of innovative drugs against Chagas disease.

Chemical Composition and Inhibitory Effects of Hypericum brasiliense and H. connatum on Prolyl Oligopeptidase and Acetylcholinesterase Activities.

BACKGROUND: The genus Hypericum (family Clusiaceae) comprises various species that are used in traditional medicine, such as wound healing, antidepressant, and anticancer agents. OBJECTIVE: The aim of this study was to evaluate the inhibitory capacity of extracts and fractions from two Hypericum species used in the Brazilian folk medicine (H. brasiliense and H. connatum) against the enzymes prolyl oligopeptidase (POP), dipeptidyl peptidase-IV (DPP-IV), and acetylcholinesterase (AChE), as well as to identify their main active constituents. METHODS: Dried aerial parts of H. connatum and H. brasiliense were subjected to extraction with 8:2 methanol-H2O. Each hydroalcoholic extract was fractioned resulting in ethyl acetate and aqueous fractions. The activity of POP, DPP-IV and AChE was determined in vitro in 96-well microplates. RESULTS: The main components identified in the plant extracts were chlorogenic acid (1), quercitrin (2), rutin (3), quercetin (4), and isoquercitrin (5). Hydroalcoholic extracts, ethyl acetate and aqueous fractions showed high POP inhibitory activity with IC50 values of 2.6 to 3.7 µg/mL. AChE and DPP-IV inhibitory effects were very low for all extracts and substances. CONCLUSION: Chlorogenic acid (1) and quercetin (4) were the main constituent responsible for the activity observed against POP. Parallel artificial membrane permeability assay of ethyl acetate fractions of both species showed that the metabolite that can effectively pass through the lipid membrane is 4, the aglycone form of 2, 3 and 5.

The components of the angiotensin-(1-7) system are differentially expressed during follicular wave in cattle.

INTRODUCTION: This study was based on the hypothesis that some components of the angiotensin-(1-7) (Ang-(1-7)) system are differentially expressed during follicular development and can be involved in the follicular health/atresia transition in bovine. MATERIAL AND METHODS: The largest (F1) and second largest follicles (F2) were collected from cows before (Day 2), during (Day 3), or after (Day 4) the expected moment of follicular deviation. In the second experiment, F1 was induced to atresia through intrafollicular injection of fulvestrant (estrogen receptor-antagonist) and, in both experiments, mRNA expression of the Mas receptor, ACE2, NEP, and PEP was evaluated in the granulosa and theca cells. RESULTS: The mRNA expression of Mas receptor was upregulated in the granulosa cells of F2 after the establishment of follicular deviation, while PEP mRNA increased during and after the deviation process. The mRNA expression of ACE2 was upregulated in the granulosa cells of F1 during and after the follicular deviation. The mRNA expression of NEP was not regulated in F1 and F2. Mas receptor expression increased in the F1 induced to atresia. CONCLUSIONS: mRNA for Mas receptor, ACE2, and PEP are differentially expressed in granulosa cells throughout follicular development and the Mas receptor can be involved with the establishment of follicular dominance.

Prolyl endopeptidase - Optimization of medium and culture conditions for enhanced production by Lactobacillus acidophilus

Background Lactic acid bacteria are able to reduce the immunoreactivity of proteins of cereal grains during wheat dough fermentation or may be a source of proteolytic preparations added during bread making. The key enzyme in prolamin degradation is prolyl endopeptidase. This study was aimed at optimizing the composition of a culture medium and culture conditions that would enhance the synthesis of intracellular prolyl endopeptidase (PEP) by Lactobacillus acidophilus 5e2. Results The application of Plackett-Burman screening plans enabled demonstrating that the concentration of a nitrogen source in the culture and the initial pH value of the culture medium were significant for PEP synthesis. Further optimization conducted with the method of central composite designs (CCD) confirmed both the linear and square impact of nitrogen concentration and initial pH value of the culture medium on PEP production. In turn, the response surface method (RSM) allowed determining the optimal nitrogen concentration and pH value at 26.88 g/l and pH 4.85, respectively. Conclusions Validation of the resultant model enabled over 3-fold increase in the quantity of the synthesized enzyme.

P-I class metalloproteinase from Bothrops moojeni venom is a post-proline cleaving peptidase with kininogenase activity: insights into substrate selectivity and kinetic behavior.

Snake venom metalloproteinases (SVMPs) belonging to P-I class are able to hydrolyze extracellular matrix proteins and coagulation factors triggering local and systemic reactions by multiple molecular mechanisms that are not fully understood. BmooMPα-I, a P-I class SMVP from Bothrops moojeni venom, was active upon neuro- and vaso-active peptides including angiotensin I, bradykinin, neurotensin, oxytocin and substance P. Interestingly, BmooMPα-I showed a strong bias towards hydrolysis after proline residues, which is unusual for most of characterized peptidases. Moreover, the enzyme showed kininogenase activity similar to that observed in plasma and cells by kallikrein. FRET peptide assays indicated a relative promiscuity at its S2-S'2 subsites, with proline determining the scissile bond. This unusual post-proline cleaving activity was confirmed by the efficient hydrolysis of the synthetic combinatorial library MCA-GXXPXXQ-EDDnp, described as resistant for canonical peptidases, only after Pro residues. Structural analysis of the tripeptide LPL complexed with BmooMPα-I, generated by molecular dynamics simulations, assisted in defining the subsites and provided the structural basis for subsite preferences such as the restriction of basic residues at the S2 subsite due to repulsive electrostatic effects and the steric impediment for large aliphatic or aromatic side chains at the S1 subsite. These new functional and structural findings provided a further understanding of the molecular mechanisms governing the physiological effects of this important class of enzymes in envenomation process.

In vitro evaluation of caffeoyl and cinnamoyl derivatives as potential prolyl oligopeptidase inhibitors.

A screening of the natural product chlorogenic acid, isolated from the Brazilian medicinal plant Hypericum brasiliense, caffeic acid, cinnamic acid, and p-methoxycinnamic acid, and derivatives of caffeoylquinic, caffeoyl, and cinnamoyl against the enzymes prolyl oligopeptidase and dipeptidyl peptidase IV was carried out. Caffeoylquinic, caffeoyl, and cinnamoyl derivatives were prepared using simple derivatization procedures and through coupling reactions with the amino acid proline. The dipeptidyl peptidase IV assay showed inhibitory activity of the tested compounds at a high concentration (500 µM) in the range of 81.5-7.2 %. In contrast, the derivatives methyl ester and 1,7-acetonide obtained from chlorogenic acid, and caffeic acid and its methyl ester derivative showed selectivity and activity as prolyl oligopeptidase inhibitors, with IC50 values of 3 to 14 mM.

Parasite prolyl oligopeptidases and the challenge of designing chemotherapeuticals for Chagas disease, leishmaniasis and African trypanosomiasis.

The trypanosomatids Trypanosoma cruzi, Leishmania spp. and Trypanosoma brucei spp. cause Chagas disease, leishmaniasis and human African trypanosomiasis, respectively. It is estimated that over 10 million people worldwide suffer from these neglected diseases, posing enormous social and economic problems in endemic areas. There are no vaccines to prevent these infections and chemotherapies are not adequate. This picture indicates that new chemotherapeutic agents must be developed to treat these illnesses. For this purpose, understanding the biology of the pathogenic trypanosomatid- host cell interface is fundamental for molecular and functional characterization of virulence factors that may be used as targets for the development of inhibitors to be used for effective chemotherapy. In this context, it is well known that proteases have crucial functions for both metabolism and infectivity of pathogens and are thus potential drug targets. In this regard, prolyl oligopeptidase and oligopeptidase B, both members of the S9 serine protease family, have been shown to play important roles in the interactions of pathogenic protozoa with their mammalian hosts and may thus be considered targets for drug design. This review aims to discuss structural and functional properties of these intriguing enzymes and their potential as targets for the development of drugs against Chagas disease, leishmaniasis and African trypanosomiasis.

Inhibitory effect of verbascoside isolated from Buddleja brasiliensis Jacq. ex Spreng on prolyl oligopeptidase activity.

The phenylpropanoid glycoside verbascoside [2-(3,4-dihydroxyphenylethyl)-1-O-α-L-rhamnopyranosyl-(1→3)-ß-D-(4-O-caffeyl)-glucopyranoside] (1) has been isolated as the main constituent of the crude extract of Buddleja brasiliensis Jacq. ex Spreng from Southern Brazil. The crude extract, main fractions and the compound 1 were evaluated for inhibition of the enzymes acetylcholinesterase (AChE), dipeptidyl peptidase-IV (DPP-IV) and prolyl oligopeptidase (POP). Compound 1 showed weak activity against DPP-IV with an IC(50) >> 150 µM and was inactive against AChE, with a pMIQ determined by bioautography of 9.6. In contrast, 1 displayed significant inhibition of POP in a dose-dependent manner with an IC(50) value of 1.3 ± 0.2 µM, similar to the positive control, baicalin, with a POP IC(50) of 12 ± 3 µM.

Molecular characterization and regulation of the angiotensin-converting enzyme type 2/angiotensin-(1-7)/MAS receptor axis during the ovulation process in cattle.

The objective of this study was to characterize the profiles of Ang-(1-7), MAS receptor, ACE(2), NEP and PEP during the ovulatory process in cattle. For this study, 40 synchronized cows with follicular diameter ≥ 12 mm were ovariectomized at different time-points (0, 3, 6, 12 and 24 h) after i.m. application of gonadotropin-releasing hormone (GnRH) to induce a luteinizing hormone surge. Follicular fluid was collected for measuring Ang-(1-7) by radioimmunoassay. Theca and granulosa cells were isolated from the preovulatory follicles to evaluate the gene expression of MAS receptor, ACE(2), NEP and PEP by qRT-PCR assay. Cross-contamination between theca and granulosa cells was tested by RT-PCR to detect cytochrome P450 aromatase (CYP19A1) and 17α-hydroxylase (CYP17A1) mRNA. Ang-(1-7) levels were constant until 12 h and then increased (p < 0.05) at 24 h after GnRH. Messenger RNA expression of MAS, ACE(2), NEP and PEP was detected in theca and granulosa cells at all time-points after GnRH. In granulosa cells, ACE(2), NEP and PEP were differentially expressed after GnRH treatment (p < 0.05). In conclusion, the Ang-(1-7), MAS receptor, ACE(2), NEP and PEP profiles in preovulatory follicles indicate that Ang-(1-7) plays a role in the regulation of the ovulatory process in cattle.

Prolyl oligopeptidase of Trypanosoma brucei hydrolyzes native collagen, peptide hormones and is active in the plasma of infected mice.

Proteases play important roles in many biological processes of parasites, including their host interactions. In sleeping sickness, Trypanosoma brucei proteases released into the host bloodstream could hydrolyze host factors, such as hormones, contributing to the development of the disease's symptoms. In this study, we present the identification of the T. brucei prolyl oligopeptidase gene (poptb) and the characterization of its corresponding enzyme, POP Tb. Secondary structure predictions of POP Tb show a structural composition highly similar to other POPs. Recombinant POP Tb produced in E. coli was active and highly sensitive to inhibitors of Trypanosoma cruzi POP Tc80. These inhibitors, which prevent T. cruzi entry into non-phagocytic cells, arrested growth of the T. brucei bloodstream form in a dose-dependent manner. POP Tb hydrolyzes peptide hormones containing Pro or Ala at the P1 position at a slightly alkaline pH, and also cleaves type I collagen in vitro and native collagen present in rat mesentery. Furthermore, POP Tb is released into the bloodstream of T. brucei infected mice where it remains active. These data suggest that POP Tb might contribute to the pathogenesis of sleeping sickness.

Flavonoids with prolyl oligopeptidase inhibitory activity isolated from Scutellaria racemosa Pers.

Prolyl oligopeptidase (POP) is a serine protease highly expressed in the brain that hydrolyses peptide bonds at the carboxyl terminal of prolyl residues. There is evidence that this enzyme participates in several functions of the central nervous system. Scutellaria racemosa Pers demonstrated significant and selective POP inhibition. Fractionation of the hydroalcoholic extract resulted in the isolation of four main constituents identified for the first time from S. racemosa Pers, the triterpenoid lupeol (1) and the flavonoids oroxylin A (5,7-dihydroxy-6-methoxyflavone, 2), hispidulin (4',5,7-trihydroxy-6-methoxyflavone, 3), and oroxyloside (oroxylin A 7-O-glucuronide, 4). Inhibitory assays indicated that 3 and 4 at a concentration of 100 microM inhibit 43 and 34% of total POP activity, respectively.