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We have previously demonstrated the presence of Angiotensin (Ang)-(1-7) in rat ovary homogenates and its stimulatory effect on estradiol and progesterone production. The present study was undertaken to identify the cellular localization of Ang-(1-7) and its receptor Mas in the rat ovary in the different phases of the estrous cycle. Ang-(1-7) and Mas were localized by immunohistochemistry and Mas mRNA expression was assessed by RT-PCR. Immunostaining for both Ang-(1-7) and Mas was found in all phases of the estrous cycle, particularly in the thecal and interstitial cells, as well as in regressing corpora lutea. However, granulosa cells were positive only in antral and preovulatory follicles at proestrus and estrus phases. This pattern contrasted with the distribution of the octapeptide Ang II, which was abundant in granulosa but not in theca cells. In addition, the expression of Mas mRNA was demonstrated in all estrous cycle phases. Angiotensin-converting enzyme activity did not vary between estrous cycle phases, whereas prolyl endopeptidase activity was significantly higher in diestrus and neutral endopeptidase activity was significantly higher in metestrus. These data provide the first evidence that new RAS components are dynamically expressed in the ovary across the rat estrous cycle. Further functional studies should clarify the role of Ang-(1-7) signaling through Mas receptor in the regulation of ovarian physiology.

Ubiquitous nature of prolamin proteins dubbed gluten from wheat and allied cereals imposes a major challenge in the treatment of celiac disease, an autoimmune disorder with no known treatment other than abstinence diet. Administration of hydrolytic glutenases as food supplement is an alternative to deliver the therapeutic agents directly to the small intestine, where sensitization of immune system and downstream reactions takes place. The aim of the present research was to evaluate the capacity of wheat grain to express and store hydrolytic enzymes capable of gluten detoxification. For this purpose, wheat scutellar calli were biolistically transformed to generate plants expressing a combination of glutenase genes for prolamin detoxification. Digestion of prolamins with barley endoprotease B2 (EP-HvB2) combined with Flavobacterium meningosepticum prolyl endopeptidase (PE-FmPep) or Pyrococcus furiosus prolyl endopeptidase (PE-PfuPep), significantly reduced (up to 67%) the amount of the indigestible gluten peptides of all prolamin families tested. Seven of the 168 generated lines showed inheritance of transgene to the T(2) generation. Reversed phase high performance liquid chromatography of gluten extracts under simulated gastrointestinal conditions allowed the identification of five T(2) lines that contained significantly reduced amounts of immunogenic, celiac disease-provoking gliadin peptides. These findings were complemented by the R5 ELISA test results where up to 72% reduction was observed in the content of immunogenic peptides. The developed wheat genotypes open new horizons for treating celiac disease by an intraluminal enzyme therapy without compromising their agronomical performance.

Thimet oligopeptidase (Top) is a thiol-sensitive metallopeptidase with increased concentrations in brain and reproductive tissues. Prolyl endopeptidase (Pep) is another endopeptidase with high enzymatic activity in the brain. The two neuropeptidases are involved in the degradation of gonadotropin-releasing hormone. To clarify their roles in reproductive regulation, the spotted scat top (sstop) and pep (sspep) genes were cloned and analyzed. Tissue distribution by RT-PCR showed that sstop and sspep were more highly expressed in reproduction-related tissues than in other tissues. The sstop and sspep mRNA was detected in female hypothalamuses at phase II (oocytes at the perinuclear stage), phase III (oocytes forming the vitelline vesicle) and phase IV (oocytes at lipidic and proteic vitellogenesis stages). Both sstop and sspep were highly expressed in phase III, moderately in phase IV, and the lowest in phase II. The sstop and sspep genes were down-regulated in hypothalamuses after treatment with estradiol both in vitro and in vivo. It is suggested that Sstop and Sspep play significant roles in the regulation of reproduction and these results suggested an ssTop- and ssPep-mediated positive feedback mechanism of ovarian function.

The human gut microbiome has a significant role in host physiology; however its role in gluten catabolism is debatable. Present study explores the role of human gut microbes in gluten catabolism and a native human gut microbe Cellulomonas sp. HM71 was identified. SSU rDNA analysis has described human gut microbiome structure and also confirmed the permanent residentship of Cellulomonas sp. HM71. Catabolic potential of Cellulomonas sp. HM71 to cleave antigenic gluten peptides indicates presence of candidate gene encoding biocatalytic machinery. Genome analysis has identified the presence of gene encoding S9A serine protease family—prolyl endopeptidase, with Ser591, Asp664 and His685 signature residues. Cellulomonas sp. HM71 prolyl endopeptidase activity was found optimal at pH 7.0 and 37 °C with a K(M) of 35.53 μmol and specifically cleaves at proline residue. Current study describes the gluten catabolism potential of Cellulomonas sp. HM71 depicting possible role of human gut microbes in gluten catabolism to confer resistance mechanisms for the onset of celiac diseases in populations with gluten diet. ELECTRONIC SUPPLEMENTARY MATERIAL: The online version of this article (10.1007/s12088-018-0732-2) contains supplementary material, which is available to authorized users.

Prolyl oligopeptidase (also named prolyl endopeptidase; PREP) hydrolyzes the Pro-Xaa bonds of biologically active oligopeptides on their carboxyl side. In 1987, we detected PREP activity in human cerebrospinal fluid (CSF) using highly sensitive liquid chromatography-fluorometry with succinyl-Gly-Pro-4-methyl-coumarin amide as a new synthetic substrate, and found a marked decrease in its activity in the cerebrospinal fluid (CSF) from patients with Parkinson's disease (PD) as compared with its level in control patients without neurological diseases. In 2013, Hannula et al. found co-localization of PREP with α-synuclein in the postmortem PD brain. Several recent studies also suggest that the level of PREP in the brain of PD patients may be related to dopamine (DA) cell death via promotion of α-synuclein oligomerization and that inhibitors of PREP may play a neuroprotective role in PD. Although the relationship between another family of prolyl oligopeptidase enzymes, dipeptidyl peptidase II (DPP II) and dipeptidyl peptidase IV (DPP IV), and α-synuclein in the PD brain is not yet clear, we found that the DPP II activity/DPP IV activity ratio in the CSF was significantly increased in PD patients. This review discusses the possibility of PREP as well as the DPP II/DPP IV ratio in the CSF as potential biomarkers of PD.

The impact of single- and two-step enzymatic modification of wheat flour proteins was investigated on their immunoreactivity. Modification of wheat proteins was conducted using preparations of prolyl endopeptidase, transglutaminase and peptidases synthesised by Lactobacillus acidophilus 5e2 and L. sanfranciscensis DSM20663. Hydrolysis of cross-linked proteins and cross-linking of proteins’ hydrolysates was used during two-step modification. Immunoreactivity of wheat flour proteins was decreased with the increase in environmental alkalinity after treatment with transglutaminase. These modifications resulted in changes in protein composition and average polypeptide chain length. Fractional composition of gliadins demonstrated that decreasing acidity of reaction medium is associated with more effective cross-linking of ω-gliadins. Protein hydrolysis by prolyl endopeptidase and peptidases from lactobacilli also resulted in effectively degrading of HMW-glutenins and ω-gliadins. The lowest content of the immunoreactive gliadins was observed for proteins degraded with peptidases. Immunological analysis of proteins subjected to two-step enzymatic modification also showed a reduction in the content of gliadins both in the samples of cross-linked protein hydrolysates, and in cross-linked proteins. Hydrolysis of cross-linked proteins favoured more the reduction in protein immunoreactivity than cross-linking of hydrolysed proteins. Wheat proteins undergo the most effective modifications favouring the reduction in immunoreactivity, when transglutaminase/peptidase LS and peptidase LS/transglutaminase combinations were used. Proposed methods of enzymatic reduction in gluten immunoreactivity could be used as additional step of flour modification in sourdough technology.

Fibroblast Activation Protein (FAP) is a membrane-bound serine protease whose expression is often elevated in activated fibroblasts associated with tissue remodeling in various common diseases such as cancer, arthritis and fibrosis. Like the closely related dipeptidyl peptidase DPPIV, the extracellular domain of FAP can be released into circulation as a functional enzyme, and limited studies suggest that the circulating level of FAP correlates with the degree of tissue fibrosis. Here we describe a novel homogeneous fluorescence intensity assay for circulating FAP activity based on a recently identified natural substrate, FGF21. This assay is unique in that it can effectively distinguish endopeptidase activity of FAP from that of other related enzymes such as prolyl endopeptidase (PREP) and was validated using Fap-deficient mice. Structural modeling was used to elucidate the mechanistic basis for the observed specificity in substrate recognition by FAP, but not by DPPIV or PREP. Finally, the assay was used to detect elevated FAP activity in human patients diagnosed with liver cirrhosis and to determine the effectiveness of a chemical inhibitor for FAP in mice. We propose that the assay presented here could thus be utilized for diagnosis of FAP-related pathologies and for the therapeutic development of FAP inhibitors.

The gluten content of rye sourdough during fermentation was monitored by a competitive ELISA based on the R5 antibody. Although a time-dependent decrease was found, the gluten content was not reduced below the threshold for gluten-free foods of 20 mg/kg, even after prolonged fermentation. Instead, Aspergillus niger prolyl endopeptidase (AN-PEP) extensively degraded gluten concentrations of up to 80,000 mg/kg in rye flour, rye sourdough, and sourdough starter under distinct temperatures and pH values. The enzyme did not lead to inactivation of the microorganisms in the sourdough starter. Gluten-free rye flour alone or in combination with sourdough starter was used to produce gluten-free bread, which was evaluated for its sensory properties. Whereas gluten-free sourdough bread had poor sensory attributes compared to a conventional rye bread used as reference, the replacement of sourdough by egg proteins yielded gluten-free bread comparable to the reference and with higher sensory quality than bread prepared from naturally gluten-free ingredients. Therefore, the feasibility of producing high-quality bread from originally gluten-containing cereals such as rye by means of treatment with AN-PEP has been shown. Rye products rendered gluten-free in this manner have the potential to increase the choice of high-quality gluten-free foods for celiac patients.

Prolyl endopeptidases (PEP) (EC 3.4.21.26), a family of serine proteases with the ability to hydrolyze the peptide bond on the carboxyl side of an internal proline residue, are able to degrade immunotoxic peptides responsible for celiac disease (CD), such as a 33-residue gluten peptide (33-mer). Oral administration of PEP has been suggested as a potential therapeutic approach for CD, although delivery of the enzyme to the small intestine requires intrinsic gastric stability or advanced formulation technologies. We have engineered two food-grade Lactobacillus casei strains to deliver PEP in an in vitro model of small intestine environment. One strain secretes PEP into the extracellular medium, whereas the other retains PEP in the intracellular environment. The strain that secretes PEP into the extracellular medium is the most effective to degrade the 33-mer and is resistant to simulated gastrointestinal stress. Our results suggest that in the future, after more studies and clinical trials, an engineered food-grade Lactobacillus strain may be useful as a vector for in situ production of PEP in the upper small intestine of CD patients. © 2014 Springer-Verlag., This research was supported by project 201370E094 from the CSIC. P.A. is the recipient of a fellowship from FICYT (BP09093) and B.D.R. is a beneficiary of a JAE-DOC contract (CSIC). C.K. is supported by a grant from the NIH (R01 DK 063158).

A noncompetitive synthetic inhibitor of prolyl endopeptidase benzyloxycarbonyl-methionyl-2(S)-cyanopyrrolidine (1.0 mg/kg intraperitoneally for 2 weeks) prevented the increase in activity of prolyl endopeptidase in the frontal cortex, striatum, and hypothalamus and activation of dipeptidyl peptidase IV in the frontal cortex of rats with experimental dopamine deficiency-dependent depressive syndrome caused by administration of proneurotoxin MPTP (2 weeks). Our results suggest that the antidepressive effect of prolyl endopeptidase inhibitor is at least partly related to prevention of enzyme activation in the frontal cortex. The antistress effect of this substance can be associated with prevention of enzyme activation in the hypothalamus.

Thrombin, a central factor in thrombogenesis, affects cells in the brain through protease activated receptors. Low levels of thrombin activity are neuroprotective while higher levels are deleterious, and we have therefore developed a new method for its direct quantitative measurement in brain slices following stroke. Thrombin activity was measured by a fluorescent substrate on fresh coronal slices taken from the ipsilateral and contralateral hemispheres 24–72 h following permanent right middle cerebral artery occlusion. Prolyl endopeptidase and aminopeptidases were inhibited as a critical step to insure the specificity of the assay for thrombin detection. Infarct volume was assessed using TTC staining. Thrombin activity in the right ischemic hemisphere was significantly higher compared to the contralateral hemisphere (32 ± 6 and 27 ± 10 mU/ml, mean ± SE in the two most affected slices from the ischemic hemisphere vs. 21 ± 6 and 8 ± 2 mU/ml in corresponding contralateral slices; p

Activities of prolyl endopeptidase and dipeptidyl peptidase IV in the frontal cortex, hypothalamus, nucleus accumbens, striatum, and hippocampus were measured in rats with the experimental anxious-depressive syndrome induced by treatment with a dipeptidyl peptidase IV inhibitor during the early postnatal period (days 5-18). Prolyl endopeptidase activity was elevated in the frontal cortex, hypothalamus, and nucleus accumbens. Increased activity of dipeptidyl peptidase IV was observed in the hypothalamus and striatum. Norepinephrine/serotonin reuptake inhibitor, imipramine, and noncompetitive prolyl endopeptidase inhibitor, benzyloxycarbonyl-methionyl-2(S)-cyanopyrrolidine, were shown to abolish depression-like behavior of animals in the forced swimming test. These compounds had a normalizing effect on activities of prolyl endopeptidase and dipeptidyl peptidase IV in brain structures of rats.

The effect of 8 factors [(with/without) daily mixing and moisture control, incubation time (t), temperature, ratio between dry substrate mass and bed’s cross section area (MA), inoculum size (spores/g), wheat germ content (WG), initial pH, and moisture content (M)] in the production of a prolyl endopeptidase (PEP) by Aspergillus niger ATCC 11414 in solid state fermentation (SSF) was tested. Contribution of all the factors was significant (p

Many synthetic peptide-based prolyl endopeptidase (PEP) inhibitors are depended on the acy-l-L-prolyl-pyrrolidine structure. The pyrrolidine ring at P1 and a large variety of lipophilic acyl groups at P3 have been reported to be important for potent PEP inhibitory activity. Upon exploration of a new lead compound for PEP inhibition, succinic acid was introduced at the P2 position to replace the l-proline in acyl-l-prolyl-pyrrolidine. A series of amido-succinylpyrrolidine compounds (1–9) were synthesized, and their PEP inhibitory activities were evaluated. Of these, compound 9 showed the most potent PEP inhibitory activity with IC50 of 6.4±0.6 nM.

Two new acetophenone glucosides, 2,6-dihydroxy-4-methoxyacetophenone 3-C-β-D-glucoside (1) and 2,6-dihydroxy-4-methoxyacetophenone 3-C-β-D-(6′-O-galloyl)glucoside (2), together with three known compounds, eugenin (3), 8-C-glucosylnoreugenin (4), and gallic acid (5) were isolated from the EtOAc soluble fraction of the MeOH extract of clove buds (Syzygium aromaticum). Of these, compound 2 showed significant prolyl endopeptidase inhibitory activity with IC50 value of 17.2±1.1 μM, suggesting it may have potential use against memory impairment and cognitive decline.

Davallialactone (1) and inoscavin A (2) isolated from Phellinus linteus showed significant prolyl endopeptidase competitive inhibition with IC50 values of 20.0±0.1 and 12.2±0.1 μM and K i values of 15.6±0.3 and 8.3±0.1 μM, respectively. They also exhibited strong antioxidant capacities against the ABTS radical system with EC50 values of 2.61±0.01, 13.3±0.05 μM, respectively.

The prolyl peptidases are a family of enzymes characterized by a biochemical preference for cleaving proline-containing peptides. The members of this enzyme family include prolyl endopeptidase, prolyl endopeptidase-like, dipeptidyl peptidase 4 (DPP4), DPP7, DPP8, DPP9, and fibroblast activation protein. DPP4 is the best studied member of the family, due to its role in physiological glucose tolerance, exerted through the regulation of the insulinotropic peptide glucagon-like peptide-1. While other members of the prolyl peptidase family have also been implicated in various (patho)physiological processes, the underlying peptides and pathways regulated by these enzymes are less clear. The identification of endogenous substrates of the prolyl peptidases is an important step in elucidating the molecular mechanisms of these enzymes. Here, we highlight the utility of liquid chromatography–mass spectrometry-based peptidomics to enable the discovery of endogenous prolyl peptidase substrates directly from tissues, and demonstrate the utility of this information in understanding the biochemical and physiological functions of the prolyl peptidases.

Anthranilic acid derivatives (3–5) containing the long chain components of N-stearoyl, -oleoyl, and-linoleoyl groups were synthesized for the study of prolyl endopeptidase (PEP, EC 3.4.21.26) inhibitory activity. Compounds 3–5 showed competitive PEP inhibition with IC50 values of 2.81, 4.07 and 3.64 μM, respectively, and K i values of 1.62, 4.10 and 2.77 μM, respectively.

α-Casein group of proteins makes up to 65% of the total casein and consists of αS1- casein, αS2- casein and other related proteins. Among all the proteases employed, chymotryptic peptides showed maximum inhibition for angiotensin converting enzyme (ACE). The degree of hydrolysis and release kinetics of the peptides during chymotrypsin hydrolysis was compared with biological activity and the potent peptides fractions were identified. The crude fraction obtained after 110 min of hydrolysis shows multifunctional activities, like ACE inhibition, antioxidant activity, prolyl endopeptidase inhibitory activity and antimicrobial activities. This fraction was further purified by HPLC and sequenced by mass spectra. This fraction constituted peptides with molecular weights of 1,205, 1,718 Da respectively. The sequencing of peptides by MALDI-TOF MS/MS shows sequences QKALNEINQF and TKKTKLTEEEKNRL from α-S2 casein.

The effects of a competitive prolyl endopeptidase inhibitor benzyloxycarbonyl-alanyl-proline were studied in rats with experimental dopamine deficiency-dependent depressive syndrome due to systemic administration of a proneurotoxin 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine for 14 days. The inhibitor was injected intraperitoneally 30 min before treatment with 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (2nd week of the study). This substance contributed to rapid disappearance of depressive symptoms during the recovery of behavioral activity. Our results indicate that benzyloxycarbonyl-alanyl-proline has the antidepressant properties.

Administration of a synthetic dipeptidyl peptidase IV inhibitor methionyl-2(S)-cyanopyrrolidine (1 mg/kg) to rats during the early postnatal period was followed by the development of behavioral changes in young and adult animals. The degree of anxiety in the elevated plus maze increased in treated rats at the age of 1-2 months. Depressive behavior in the forced swimming test was typical of animals aging 2-3 months. Diazepam reduced the severity of anxiety in treated rats. Melipramine had a normalizing effect on swimming behavior. A novel prolyl endopeptidase inhibitor benzyloxycarbonyl-methionyl-2(S)-cyanopyrrolidine had the antidepressant properties.

To understand the role of peptidases in seminal physiology of Crotalus durissus terrificus, intra- and inter-seasonal activity levels of acid (APA), basic (APB), puromycin-sensitive (APN-PS) and puromycin-insensitive neutral (APN-PI), cystyl (CAP), dipeptidyl-IV (DPPIV), type-1 pyroglutamyl (PAP-I) and prolyl-imino (PIP) aminopeptidases as well as prolyl endopeptidase (POP) were evaluated in soluble (SF) and/or membrane-bound (MF) fractions of semen collected from the ductus deferens of the male reproductive tract and from the posterior portion of the uterus. Seminal APB, PIP and POP were detected in SF, while other peptidases were detected in SF and MF. Only the convoluted posterior uterus in winter and autumn had semen. Relative to other examined peptidases, in general, APN-PI, APN-PS and APB activities were predominant in the semen from the uterus and throughout the year in the semen from the ductus deferens, suggesting their great relevance in the seminal physiology of C. d. terrificus. The levels of peptidase activities in the ductus deferens semen varied seasonally and were different from those of semen in the uterus, suggesting that their modulatory actions on susceptible peptides are integrated to the male reproductive cycle events and spermatozoa viability of this snake.

A novel inhibitor of prolyl endopeptidase, the propeptin analog propeptin-2 (C105H130N24O(24)), missing two amino acid residues from the propeptin C-terminus was isolated from the fermentation broth of propeptin-producing Microbispora sp. SNA-115 grown using a large inoculum. It shows the same enzyme inhibition activity as propeptin against prolyl endopeptidase (Ki=1.5 microM), but its antimicrobial activity against Mycobacterium phlei is more than 100-fold lower.

Prolyl endopeptidase (PEP, EC 3.4.21.26), a serine protease, is widely distributed in various organs, particularly in the brains of Alzheimer's disease patients. The expression of PEP in Alzheimer's patients has been found to be significantly higher than that of the normal person, suggesting that a specific PEP inhibitor can be a good candidate for an anti-amnestic drug. In the current study, thirty-nine plant phenolics were investigated to determine their roles as prolyl endopeptidase (PEP) inhibitors. Nineteen compounds such as 1,2,3-trigalloyl glucopyranoside, 1,2,6-trigalloyl glucopyranoside, 1,2,3,4,6-pentagalloyl gluco-pyranoside, 1,2,6-trigalloyl alloside, 1,3,6-trigalloyl alloside, 1,2,3,6-tetragalloyl alloside, acetonyl geraniin, corilagin, elaeocarpusin, euphorscopin, geraniin, helioscopin B, helioscopinin A, helioscopinin B, jolkinin, macranganin, rugosin E, supinanin, and teracatain exhibited strong inhibition against PEP (IC50 26.7 - 443.7 x 10(-9) M). Rugosin E (IC50 26.7 x 10(-9) M) showed the most effective inhibition followed by 1,2,6-trigalloyl glucopyranoside (IC5031.4 x 10(-9) M) and macranganin (IC5042.6 x 10(-9) M). No significant structure-activity relationship was found; however, at least, three pyrogallol groups seem to be a minimal requirement for stronger activity against PEP All 19 active compounds inhibited PEP in a non-competitive mode with a substrate in Dixon plots. They did not show significant effects against other serine proteases such as trypsin, chymotrypsin and elastase, indicating that they were relatively specific PEP inhibitors.

Prolyl endopeptidase (PEP) is believed to inactivate neuropeptides that are present in the extracellular space. However, the intracellular localization of PEP suggests additional, yet unidentified physiological functions for this enzyme. Here we studied the expression, enzymatic activity and subcellular localization of PEP in adult and aged mouse brain as well as in brains of age-matched APP transgenic Tg2576 mice and in brains of Alzheimer's disease patients. In mouse brain PEP was exclusively expressed by neurons and displayed region- and age-specific differences in expression levels, with the highest PEP activity being present in cerebellum and a significant increase in hippocampal but not cortical or cerebellar PEP activity in aged mouse brain. In brains of young APP transgenic Tg2576 mice, hippocampal PEP activity was increased compared to wild-type littermates in the pre-plaque phase but not in aged mice with beta-amyloid plaque pathology. This "accelerated aging" with regard to hippocampal PEP expression in young APP transgenic mice might be one factor contributing to the observed cognitive deficits in these mice in the pre-plaque phase and could also explain in part the cognition-enhancing effects of PEP inhibitors in several experimental paradigms.

A number of neuropeptides are affected in Parkinson's disease and the enzyme proline endopeptidase contributes to the degradation of many of these neuropeptides, some of which are linked to a variety of cognitive functions. In the present study, the effects of the highly potent proline endopeptidase inhibitor S 17092 on cognitive deficits in monkeys induced by chronic low dose 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) administration were examined. Chronic low dose MPTP administration resulted in deficits in performance of variable delayed response, delayed matching-to-sample, and delayed alternation tasks. Seven day oral administration of S 17092 followed by single dose administration of the same dose on the day of testing significantly improved overall performance on these tasks. The most effective dose of S 17092 was 3 mg/kg. These results indicate that S 17092 has cognition-enhancing properties in this model of early parkinsonism.

Three prolyl endopeptidase (PEP) inhibitors were isolated from the methanolic extract of green tea leaves. They were identified as (-)-epigallocatechin gallate, (-)-epicatechin gallate, and (+)-gallocatechin gallate with the IC50 values of 1.42 x 10(-4) mM, 1.02 x 10(-2) mM, and 1.09 x 10(-4) mM, respectively. They were non-competitive with a substrate in Dixon plots and did not show any significant effects against other serine proteases such as elastase, trypsin, and chymotrypsin, suggesting that they were relatively specific inhibitors against PEP. The isolated compounds are expected to be useful for preventing and curing of Alzheimer's disease.

Angiotensin II is recognised as the principle active peptide of the renin-angiotensin system, exerting effects on fluid and electrolyte homeostasis, and cardiovascular control including neural and long term trophic effects. However, recent studies indicate that other angiotensin peptides such as angiotensin III, angiotensin II (1-7) and angiotensin IV, may have specific actions. Interestingly, recent work involving angiotensin IV demonstrates that this peptide binds to specific receptors and may be involved in memory retention and neuronal development. Furthermore, our demonstration that a globin fragment, LVV-haemorphin-7, binds with high affinity to the angiotensin IV binding site and is abundant in the brain, indicates that this may represent a novel brain neuropeptide system. It now appears, that the renin-angiotensin system is more complex than previously thought and capable of generating multiple, active peptides which elicit numerous diverse actions.

The effect of a novel prolyl endopeptidase (PEP) inhibitor, (S)-2-[[(S)-2-(hydroxyacetyl)-1-pyrrolidinyl] carbonyl]-N-(phenylmethyl)-1-pyrrolidine-carboxamide (JTP-4819), on neuropeptide metabolism was investigated in the rat brain. JTP-4819 exhibited a strong in vitro inhibitory effect on cortical and hippocampal PEP activity, with the IC50 values being approximately 0.58 +/- 0.02 and 0.61 +/- 0.06 nM, respectively. JTP-4819 also inhibited the in vitro degradation of substance P (SP), arginine-vasopressin (AVP), and thyrotropin-releasing hormone (TRH) by rat brain supernatants, with the IC50 values being respectively 3.4, 2.1, and 1.4 nM in the cerebral cortex and 3.3, 2.8, and 1.9 nM in the hippocampus. Oral administration of JTP-4819 at doses of 1 and 3 mg/kg increased SP-like immunoreactivity (LI) and AVP-LI in the cerebral cortex. JTP-4819 also increased hippocampal SP-LI and AVP-LI at doses of 1 and 3 mg/kg, as well as hippocampal TRH-LI at a dose of 3 mg/kg. These findings suggest that JTP-4819 inhibited the degradation of SP, AVP, and TRH in the rat brain secondary to the inhibition of PEP, and thus increased cortical and hippocampal SP-LI and AVP-LI as well as hippocampal TRH-LI.

Background Chronic obstructive pulmonary disease (COPD) is a common respiratory disorder for which new diagnostic and therapeutic approaches are required. Hallmarks of COPD are matrix destruction and neutrophilic airway inflammation in the lung. We have previously described two tri-peptides, N-α-PGP and PGP, which are collagen fragments and neutrophil chemoattractants. In this study, we investigate if N-α-PGP and PGP are biomarkers and potential therapeutic targets for COPD. Methods Induced sputum samples from COPD patients, healthy controls and asthmatics were examined for levels of N-α-PGP and PGP using mass spectrometry and for the ability to generate PGP de novo from collagen. Proteases important in PGP generation in the lung were identified by the use of specific inhibitors in the PGP generation assay and by instillation of proteases into mouse lungs. Serum levels of PGP were compared between COPD patients and controls. Results N-α-PGP was detected in most COPD sputum samples but in no asthmatics or controls. PGP was detected in a few controls and in all COPD sputum samples, where it correlated with levels of myeloperoxidase. COPD sputum samples had the ability to generate N-α-PGP and PGP de novo from collagen. PGP generation by COPD sputum was blocked by inhibitors of matrix metalloproteases (MMP's) 1 and 9 and prolyl endopeptidase. MMP's 1 and 9 and prolyl endopeptidase acted synergistically to generate PGP in vivo when instilled into mouse lungs. Serum levels of PGP were also significantly higher in COPD patients than in controls Conclusion N-α-PGP and PGP may represent novel diagnostic tests and biomarkers for COPD. Inhibition of this pathway may provide novel therapies for COPD directed at the chronic, neutrophilic, airway inflammation which underlies disease progression.

The activities of dipeptidyl peptidase IV (DPP-IV), prolyl endopeptidase (PE) and cathepsin B (CB) were investigated in primary human lung tumours and matched lung parenchyma, using continuous-rate fluorometric assays of the enzymes. Squamous-cell lung carcinomas showed significantly higher specific activities of all three enzymes studied. In lung adenocarcinomas only activities of PE and CB were increased significantly. In a limited number of primary human lung tumours of other histological types the activities of DPP-IV, PE and CB were also elevated. Mixing the matched homogenates of lung tumours and lung parenchyma gave additive activities for each enzyme studied. A significant correlation between tumour/lung ratios of specific activities of DPP-IV and CB was observed.

Proline-specific endopeptidase (PSE) (EC 3.4.21.26) from Flavobacterium meningosepticum was subjected to partial amino acid sequencing. According to the peptide sequences obtained, oligonucleotides were used to amplify a PSE-specific DNA fragment of 930 bp from F. meningosepticum genomic DNA, employing the polymerase chain reaction technique. This fragment served as a molecular probe to isolate the respective gene. DNA sequencing revealed that the PSE gene consists of 2118 bp coding for a 78,634 Da protein of 705 amino acids. The coding region was cloned in different expression vectors of Escherichia coli. Transformed E. coli cells overproduce an active prolyl endopeptidase of 75,000 relative molecular mass, which is delivered to the bacterial periplasmic space. Up to 1.6 units of active prolyl endopeptidase were obtained from 1 mg E. coli cells. Furthermore, the efficient purification of active prolyl endopeptidase from the periplasm of recombinant E. coli cells is described.

Background In 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 (α-MSH), we developed a new mass spectrometry based technique to quantitate, in multiplex, the various forms of α-MSH. Methods Using Multiple Reaction Monitoring (MRM), we analyzed peptide transitions to quantify three different forms of α-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. Results We first demonstrate in vitro that PREP digests biological active alpha melanocyte stimulating hormone (α-MSH1–13), by cleaving the terminal amidated valine and releasing a truncated alpha melanocyte stimulating hormone (α-MSH1–12) product – the 12 residues α-MSH form. We then use the technique in vivo to analyze the MRM transitions of the three different forms of α-MSH: the deacetylated α-MSH1–13, the acetylated α-MSH1–13 and the truncated form α-MSH1–12. 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 α-MSH1–13 and α-MSH1–12 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 α-MSH1–13 versus the deacetylated α-MSH1–13. These results combined with the demonstration that PREP digests α-MSH1–13 in vitro, strongly suggest that α-MSH1–13 is an in vivo substrate of PREP. Conclusion The 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.

International audience; Thyrotropin-Releasing hormone (TRH)-degrading pyroglutamyl peptidase I (PGP I) and prolylendopeptidase (PE) activities have been demonstrated in rat insulinoma RINm 5F cell line. These two enzymes catalyze the conversion of TRH to Histydyl-Proline-Diketopiperazine and to acid TRH respectively. After cell fractionation, we found all the PGP I and PE activities in the cytosolic fraction. The membrane-bound PGP II activity is not detectable in the RINm 5F cells. Further investigations on these two cytosolic enzymes show that pyroglutamyl- and proline-containing peptides are inhibitors of each TRH-degrading enzyme. Gel filtration chromatography on Sephadex G100 shows that PGP I and PE activity have an apparent molecular mass of about 18 kDa and 57 kDa, respectively. Kinetic analysis with TRH as substrate, gives a Km of 44 microM and 235 microM, and a Vmax of 1.49 and 8.80 pmol/min/micrograms protein for PGP I and PE, respectively. Immunoreactive TRH, His-Pro-Diketopiperazine and acid TRH levels in the cell line extracts are 2.2 +/- 0.9, 22.5 +/- 11.1 and 28.7 +/- 14.6 pg/1O6 cells, respectively. When cells have been incubated for 2 to 72 hours with a P.E. inhibitor (Z-Gly-Pro-CHN2) at 5 x 10(-7) M, both cell PGP I and PE activities are inhibited. No change in the cellular content of immunoreactive TRH, His-Pro-Diketopiperazine and acid TRH have been observed in treated cells.(ABSTRACT TRUNCATED AT 250 WORDS)

Researchers are utilising a variety of approaches in the quest for more effective treatments for neurodegenerative diseases and CNS disorders. Of particular note at the 13th International Symposium on Medicinal Chemistry [ Paris, France; September 1994 ] were new muscarinic agonists and prolyl endopeptidase inhibitors for dementia, and a novel anxiolytic from Servier. The idea of using melatonin agonists to control sleep disturbances was also discussed, with evidence of potential benefit provided by animal studies of S-20098.

Inhibitory effects on fertilization of the ascidian of three benzyloxycarbonyl(Z)-aminoacyl prolinals and Z-Gly-Pro-chloromethyl ketone added before and after insemination were examined. The results suggest that the prolyl endopeptidase is involved in the process of fertilization, especially in a process taking place between chorion elevation and cell cleavage.