Ontogeny of prolyl endopeptidase and pyroglutamyl peptidase I in rat tissues

Prolyl endopeptidase and pyroglutamyl peptidase I are enzymes which participate in the degradation of thyrotropin-releasing hormone (TRH), a hormone which is thought to play an important role in the development of organs and tissues. Here, we have characterized the ontogeny of TRH degrading enzyme activity in the brain cortex, lung, heart, kidney and liver. Overall, prolyl endopeptidase activity was found to be 2 to 5 fold higher in newborn vs. adult rat tissues, with the exception of the soluble form in the liver and the particulate form in the lung. In contrast, the developmental profile of pyroglutamyl peptidase I activity was found to be more variable and tissue dependent. These results corroborate the idea that both enzymes play important, tissue-specific roles during the development and maturation of rat organs.     

Specificity of prolyl endopeptidase

A series of tetrapeptides, Cbz(Bz)-Gly-X-Leu-Gly, were synthesized and the kinetic parameters, kcat and kcat/Km, determined for their hydrolyses by prolyl endopeptidase from Flavobacterium. The peptides with X = N-Me-Ala, Sar and Ala as well as the standard substrate (X = Pro) were found to be good substrates, while those with X = alpha-aminobutyryl, Hyp, Ser and Gly were poor substrates, and those with X = pipecolyl, alpha-aminoisobutyryl, N-Me-Val, N-Me-Leu, Hyp(O-Bzl) and Ser(O-Bzl) were not cleaved at all. These results suggest that the specificity-determining site or S1 subsite of the enzyme is designed to fit exactly the proline residue of the substrate with allowance for the residues carrying substituents at the N and/or C alpha which must not exceed the size of the pyrrolidine ring of proline.     

Distribution of prolyl endopeptidase activities in rat and human brain.

Prolyl endopeptidase is a proteolytic enzyme which could have a neuropeptide catabolising role in the central nervous system. Although prolyl endopeptidase has been described as a cytosolic enzyme, it has become clear that it can also be found in particulate form. The regional and subcellular distribution of this enzyme was evaluated in rat and human brain. The activity of the enzyme was higher in the human than in the rat brain. In the human brain, the activity levels of both soluble and particulate prolyl endopeptidase were the highest in frontal, parietal and occipital cortices and the lowest in the cerebellum. In the rat brain, the regional distribution of the enzyme was more homogeneous. The activity in all the areas of the central nervous system is higher than in peripheral tissues. Subcellular distribution of the enzyme in the brain indicates that prolyl endopeptidase was higher in the cytosolic fraction than in the particulate fractions. The particulate form was enriched in the synaptosomal and the myelinic membranes. The high activity of prolyl endopeptidase in the human cortex suggests that prolyl endopeptidase could play a role in the functions of this brain area.     

Regulation of prolyl endopeptidase activity by the intracellular redox state

The activity of prolyl endopeptidase was markedly decreased during incubation of intact murine erythroleukemia cells at 45 degrees C, but not during incubation of sonicated cells or during incubation at 42 degrees C. The thermal inactivation of prolyl endopeptidase in situ required neither the synthesis of proteins and polynucleotides nor the synergistic activation of inhibitors. Moreover, inhibition of lysosomal proteinases and calpains or depletion of ATP did not affect the thermal inactivation of prolyl endopeptidase. This specific inactivation of prolyl endopeptidase was also observed following the addition to the culture medium of menadione or diamide, compounds known to increase intracellular oxidized glutathione levels. The activity of prolyl endopeptidase in the cell lysate was also dose-dependently decreased by the addition of glutathione disulfide and the decrease of the activity was prevented by coexistence of reduced glutathione. Furthermore, the level of intracellular oxidized glutathione was increased during incubation at 45 degrees C for 15 min, but not at 42 degrees C for 30 min. These results strongly suggest that the activity of prolyl endopeptidase is regulated by changes in the intracellular redox potential.     

Screening of crude drug extracts for prolyl endopeptidase inhibitory activity.

Prolyl endopeptidase (PEP, EC 3.4.21.26) is an enzyme to play a role in metabolism of proline-containing neuropeptides, such as vasopressin, substance P and thyrotropin-releasing hormone (TRH), which were suggested to be involved with learning and memory processes. Then, specific inhibitor of PEP is expected to have antiamnesic effects, and thus we screened forty-six water- and methanol-extracts from crude drugs selected on the basis of traditional Chinese medicine theory, for Flavobacterium prolyl endopeptidase inhibition. Among them, the water-extracts of Rhodiola sacra (IC50, 0.77 microgram/ml) and the methanol-extracts of Lycopodium clavatum (IC50, 1.3 micrograms/ml), Paeonia lactiflora var. trichocarpa (IC50, 5.7 micrograms/ml), Paeonia veitchii (IC50, 2.4 micrograms/ml) and Rhodiola sacra (IC50, 0.67 microgram/ml) showed strong inhibitory activity. In addition, we also examined the PEP inhibitory activity of eleven compounds from Salvia deserta, and found that in addition to a catechol group alpha-hydroxy-para-quinone group may be related to the PEP inhibition.     

Characterization of membrane-bound prolyl endopeptidase from brain

Prolyl oligopeptidase (POP) is a serine protease that cleaves small peptides at the carboxyl side of an internal proline residue. Substance P, arginine-vasopressin, thyroliberin and gonadoliberin are proposed physiological substrates of this protease. POP has been implicated in a variety of brain processes, including learning, memory, and mood regulation, as well as in pathologies such as neurodegeneration, hypertension, and psychiatric disorders. Although POP has been considered to be a soluble cytoplasmic peptidase, significant levels of activity have been detected in membranes and in extracellular fluids such as serum, cerebrospinal fluid, seminal fluid, and urine, suggesting the existence of noncytoplasmic forms. Furthermore, a closely associated membrane prolyl endopeptidase (PE) activity has been previously detected in synaptosomes and shown to be different from the cytoplasmic POP activity. Here we isolated, purified and characterized this membrane-bound PE, herein referred to as mPOP. Although, when attached to membranes, mPOP presents certain features that distinguish it from the classical POP, our results indicate that this protein has the same amino acid sequence as POP except for the possible addition of a hydrophobic membrane anchor. The kinetic properties of detergent-soluble mPOP are fully comparable to those of POP; however, when attached to the membranes in its natural conformation, mPOP is significantly less active and, moreover, it migrates anomalously in SDS/PAGE. Our results are the first to show that membrane-bound and cytoplasmic POP are encoded by variants of the same gene.     

Pyroglutamyl peptidase I and prolyl endopeptidase in human semen: increased activity in necrozoospermia

Thyrotropin-releasing hormone (TRH) and its analogues have been reported to have important functions in human semen. In the present paper, we have characterized the activity of the TRH-degrading enzymes pyroglutamyl peptidase I and prolyl endopeptidase in the fluid and prostasomes of human semen and in subcellular fractions of the corresponding sperm. Enzymatic activities were measured fluorimetrically using beta-naphthylamine derivatives as substrate. Activity associated with both enzymes was detected in seminal fluid and in the prostasome fraction, as well as in soluble and particulate sperm subcellular fractions. Pyroglutamyl-peptidase I activity presented highest levels in the particulate sperm fraction, whereas the activity of prolyl endopeptidase was maximal in the soluble sperm fraction. In addition, we compared the activity of both enzymes in different seminal fractions in normozoospermic, fertile men and in subfertile patients with different abnormalities revealed by spermiogram analysis (astenozoospermia, necrozoospermia and teratozoospermia). The activities of pyroglutamyl peptidase I and prolyl endopeptidase in necrozoospermia were found to be higher in the corresponding soluble and particulate sperm fractions, respectively, with respect to those measured in normozoospermic semen. The results of the present study indicate that these enzymes may participate in regulating the levels of seminal TRH analogues and in mediating sperm death associated with necrozoospermia.     

The effect of inhibitors of prolyl endopeptidase and pyroglutamyl peptide hydrolase on TRH degradation in rat serum

The identity of the enzymes catalyzing the degradation of thyrotropin releasing hormone (TRH) in rat serum was investigated by the use of specific inhibitors of prolyl endopeptidase and pyroglutamyl peptide hydrolase. These inhibitors did not protect TRH from degradation, but o-phenanthroline afforded significant protection. The participation of "thyroliberinase", a metalloenzyme which cleaves TRH at the pyroglutamyl-His bond was implied. A coupled assay using the chromogenic substrate pyroglutamyl-His-Pro-2-naphthylamide and excess diaminopeptidase IV was developed to specifically quantitate "thyroliberinase" activity. Rat serum catalyzed the degradation of 67.5 nmoles substrate/ml serum/h. The data indicate that TRH is degraded in rat serum predominantly by "thyroliberinase" and that prolyl endopeptidase and pyroglutamyl peptide hydrolase do not contribute significantly to this process.     

Presence of an endopeptidase activity in rat liver ribosomes

Preparation of ribosomes using different procedures (treatment of postmitochondrial-postlysosomal supernatant or microsomes with 1% triton in 0.15 or 0.5 M KCl and subsequent sucrose gradient centrifugation; treatment of microsomes with 1.5% deoxycholate/2% triton) results in purified ribosomes which contain an endopeptidase activity detectable by breakdown of ribosomal proteins to trichloroacetic acid soluble split products. The proteolytic activity can be recovered also in the extracted proteins of whole ribosomes. With ribosomes the pH optimum of proteolytic breakdown is at about 7. The inhibition of the activity by leupeptin, DIFP and soya bean trypsin inhibitor suggests a serine type of the proteolytic activity.     

Murine T cells expressing high activity of prolyl endopeptidase are susceptible to activation-induced cell death

Prolyl endopeptidase (PEP) is widely distributed and thought to play an important role in the degradation of peptide hormones and neuropeptides, but its biological role is totally unknown. In this study, we examined PEP activity in subpopulations of murine T cells and found that PEP activity was significantly higher in immature thymocytes than in mature thymocytes or in peripheral T cells. Stimulation of murine peripheral T cells time-dependently increased PEP activity. Although murine T cell hybridomas exhibited high PEP activity, the PEP activity was fully inhibited by treatment with PEP inhibitor. The pretreated T cells were found to be resistant to activation-induced cell death (AICD). Similar results were obtained in murine thymocytes as well as in activated peripheral T cells. PEP activity in T cell hybridomas remained unchanged during AICD. These results suggest that T cells expressing high PEP activity are susceptible to ACID.     

Slow tight-binding inhibition of prolyl endopeptidase by benzyloxycarbonyl-prolyl-prolinal.

Prolyl endopeptidase is a serine proteinase that specifically cleaves peptides on the carboxy side of proline residues. Wilk & Orlowski [(1983) J. Neurochem. 41, 69-75] have shown that benzyloxycarbonyl-prolyl-prolinal (Z-prolyl-prolinal) is a potent inhibitor of prolyl endopeptidase. We show that Z-prolyl-prolinal is a slow-binding inhibitor of mouse brain prolyl endopeptidase with Ki 0.35 +/- 0.05 nM. Kinetic analysis indicates that the mechanism is a simple, but slow, reversible equilibrium between free and bound enzyme (E + I in equilibrium EI) with rate constants for association (kon) and dissociation (koff) of 1.6 X 10(5) M-1.s-1 and approx. 4 X 10(-5) s-1 respectively. Slow-binding inhibition is dependent on the presence of the aldehyde group since the alcohol (Z-prolyl-prolinol) is a rapid and 50,000-fold poorer inhibitor (Ki 19 microM). Prolyl endopeptidase from human brain is also inhibited by Z-prolyl-prolinal with kinetics similar to those of the mouse brain enzyme.     

Directed evolution to improve the thermostability of prolyl endopeptidase

Prolyl endopeptidase is the only endopeptidase that specifically cleaves peptides at proline residues. Although this unique specificity is advantageous for application in protein chemistry, the stability of the enzyme is lower than those of commonly used peptidases such as subtilisin and trypsin. Therefore, we attempted to apply a directed evolution system to improve the thermostability of the enzyme. First, an efficient expression system for the enzyme in Escherichia coli was established using the prolyl endopeptidase gene from Flavobacterium meningosepticum. Then, a method for screening thermostable variants was developed by combining heat treatment with active staining on membrane filters. Random mutagenesis by error-prone PCR and screening was repeated three times, and as a result the thermostability of the enzyme was increased step by step as the amino acid substitutions accumulated. The most thermostable mutant obtained after the third cycle, PEP-407, showed a half-life of 42 min at 60 degrees C, which was 60 times longer than that of the wild-type enzyme. The thermostable mutant was also more stable with a high concentration of glycerol, which is a necessary condition for in vitro amidation.     

Cadmium‐induced toxicity increases prolyl endopeptidase (PREP) expression in the rat testis

During the differentiation of the male gamete, there is a massive remodeling in the shape and architecture of all the cells of the seminiferous epithelium. The cytoskeleton, as well as many associated proteins with it, plays a pivotal role in this process. The testis is particularly susceptible to environmental pollutant, which can lead to injury and impairment of normal spermatozoa production. Cadmium (Cd) is one of the major chemical environmental toxicants in economically developed countries. Food and cigarettes are the main sources of exposure to this element. Here, the protective role of zinc (Zn) to prevent the testicular toxicity in male adult rats after prenatal and during lactation exposure to Cd has been assessed. Altered testicular histology at the interstitial and germinal levels was found, whereas Zn supply completely corrected Cd toxicity. Moreover, the effects of these metals on the testicular expression and localization of the protease prolyl endopeptidase (PREP) were evaluated. Interestingly, the results showed an increase of PREP messenger RNA and protein. Data were corroborated by immunofluorescence. This study raises the possibility of using PREP as a new fertility marker.     

Purification and characterization of prolyl endopeptidase from pig brain

The prolyl endopeptidase from pig brain was purified to homogeneity according to SDS-gel electrophoresis and visualization with the silver staining procedure. The molecular weight of prolyl endopeptidase was estimated as 70 kDa, and the isoelectric point as 4.9. The molecular properties of prolyl endopeptidase from pig brain are therefore similar to those of prolyl endopeptidases from other mammalian tissues. Diisopropylfluorophosphate, diethylpyrocarbonate and p-chloromercuribenzoic acid are strong irreversible inhibitors of prolyl endopeptidase from pig brain. We showed that diisopropylfluorophosphate und diethylpyrocarbonate act as competitive inhibitors with respect to substrate. Therefore it is assumed that at least one serine and one histidine residue are located at the active site of this enzyme. This result supports the assumption that the prolyl endopeptidase from pig brain is a typical serine protease. Substance P, thyreoliberin, beta-casomorphin-5 and morphiceptin are hydrolysed by prolyl endopeptidase in vitro.     

pH-dependent mechanism in the catalysis of prolyl endopeptidase from pig muscle

Prolyl endopeptidase, an enzyme exhibiting high specificity towards the Pro-Xaa bond, is thought to play an important role in the metabolism of biologically active peptides. (a) It has been found that pig muscle is an appropriate source for the preparation of a reasonable quantity of enzyme. Thus, 1 kg muscle yields 1-1.5 mg enzyme, homogeneous by fast protein liquid chromatography. (b) Bulky reagents reacting with cysteine residues inactivate the enzyme almost completely, whereas the small molecule iodoacetamide only partially inhibits the catalytic activity. This indicates that the thiol group is not essential for catalysis, but it is located at or near the active site. (c) Kinetic analysis has indicated that prolyl endopeptidase is remarkably sensitive to ionic strength. Addition of salts, e.g. sodium chloride, to the reaction mixture, up to 0.5 M, considerably enhances the rate of acylation, suggesting that charged groups of the protein exert significant effects on the catalytic site. (d) This is supported by the doubly sigmoidal character of the pH-rate profile. The existence of the doubly sigmoidal curve also indicates that the enzyme has two forms, which exhibit different activities and interconvert with changing pH. (e) The low-pH form displays a significant kinetic deuterium isotopic effect (1.70 and 1.89 in the absence and in the presence of 0.5 M NaCl, respectively), as usually observed in the serine protease catalysis. In contrast, the high-pH form, which is physiologically operative, however, has practically no kinetic deuterium effect (1.09 and 1.15 in the absence and in the presence of 0.5 M NaCl, respectively). It is concluded that a general base/acid-catalyzed acylation step is rate-limiting in the lower pH range, and an isotopically silent step, probably a conformational change preceding acylation, dominates the reaction in the physiological pH range.     

Effects of L-DOPA administration upon monoamine oxidase activity in rat tissues

The effects of L-DOPA administration in various doses (250,500 and 1000 mg/kg for 7 days) upon type A and B MAO activities in rat tissues have been investigated using the substrates 5-HT, tyramine and benzylamine. The specific activities of MAO in heart, kidney and brain were significantly increased after L-DOPA, whereas liver and vas deferens MAO was unchanged. None of the observed changes was totally specific for either form of the enzyme, although some evidence for a slight selectivity on type A MAO of heart and type B MAO of kidney and brain was obtained. These results indicate that some tissues may respond to elevated intracellular catecholamine levels, resulting from L-DOPA administration, by increasing their capacity to deaminate those amines. However, at present, the exact mechanism by which these changes are brought about is unknown.     

Subcellular localization suggests novel functions for prolyl endopeptidase in protein secretion

For a long time, prolyl endopeptidase (PEP) was believed to inactivate neuropeptides in the extracellular space. However, reports on the intracellular activity of PEP suggest additional, as yet unidentified, physiological functions for this enzyme. Here, we demonstrate using biochemical methods of subcellular fractionation, immunocytochemical double-labelling procedures and localization of PEP-enhanced green fluorescent protein fusion proteins that PEP is mainly localized to the perinuclear space, and is associated with the microtubulin cytoskeleton in human neuroblastoma and glioma cell lines. Disassembly of the microtubules by nocodazole treatment disrupts both the fibrillar tubulin and PEP labelling. Furthermore, in a two-hybrid screen, PEP was identified as binding partner of tubulin. These findings indicate novel functions for PEP in axonal transport and/or protein secretion. Indeed, a metabolic labelling approach revealed that both PEP inhibition and PEP antisense mRNA expression result in enhanced peptide/protein secretion from human U-343 glioma cells. Because disturbances in intracellular transport and protein secretion mechanisms are associated with a number of ageing-associated neurodegenerative diseases, cell-permeable PEP inhibitors may be useful for the application in a variety of related clinical conditions.     

Localization of prolyl endopeptidase mRNA in small growing follicles of porcine ovary

Prolyl endopeptidase (EC3.4.21.26) has been considered a unique intracellular enzyme catalyzing internal peptide bond hydrolysis of Pro-X. In this study, the distribution of prolyl endopeptidase activity and its mRNA was investigated in the follicles of porcine ovary. Both follicular fluid and granulosa cell fractions from small follicles showed higher activity than those from large follicles. Molecular cloning and Northern blot analysis suggested that only one species of prolyl endopeptidase gene was expressed in the ovary. In addition, in situ hybridization study revealed that the prolyl endopeptidase mRNA expresssion was more noticeable in the granulosa cell layers of small ovarian follicles than in those of large follicles, suggesting its importance in the early stage of follicular development. Mol. Reprod. Dev. 50:121–127, 1998. © 1998 Wiley-Liss, Inc.     

A novel leupeptin-sensitive serine endopeptidase present in normal and malignant rat mammary tissues

Summary N-Methyl-N-nitrosourea (MNU)-induced rat mammary adenocarcinomas contain high levels of a novel leupeptin-sensitive serine endopeptidase. Its properties apparently differ from those of other similar endopeptidases reported to be present in various normal and malignant mammalian tissues. The same leupeptinsensitive serine endopeptidase was also detected in normal rat mammary tissues, but at levels approximately 20 times lower than those in MNU-induced mammary tumors.This enzyme, which is a trypsin-like serine endopeptidase, preferentially hydrolyzes various synthetic endopeptidase substrates at the carboxyl side of an arginyl residue. It has an apparent Mr of approximately 160,000 and a Stokes radius of 49\rA, as determined by gel filtration. Its isoelectric points range from 4.5 to 4.8, and it has a pH optimum of approximately 7.0. The enzyme is stable from pH 4.0 to 7.0, but is extremely unstable above pH 7.0. Besides leupeptin, its activity is inhibited by antipain, aprotinin, N-p-tosyl-L-lysine chloromethyl ketone and phenylmethylsulfonyl fluoride, but is not inhibited by soybean trypsin inhibitor. Many other potential inhibitors or activators such as 2-mercaptoethanol, p-hydroxymercuribenzoic acid and EDTA have no effect on its activity. The enzyme is adsorbed to p-aminobenzamidine agarose affinity beads at pH 6.5 and elutes at pH 4.0.Abbreviations BAPA -N-benzoyl-DL-arginine-4-nitroanilide (all other optically active amino acids in the substrates or inhibitors are of the L-configuration except for the valyl residue in DL-Val-Leu-Arg-4NA) - 4NA 4-nitroanilide - Bz -N-benzoyl - Cbz -N-benzyloxycarbonyl - Suc -N-succinyl - Tos p-toluenesulfonyl - CH₂Cl chloromethyl ketone - DMBA 7,12-dimethylbenz(a)anthracene - MNU N-methyl-Nnitrosourea - Mr apparent relative molecular mass