Prolyl Endopeptidase Activity Research Articles

Higher serum prolyl endopeptidase activity in patients with post-traumatic stress disorder

Background: It is reported that psychiatric disorders, such as depression and schizophrenia, are associated with changes in serum activity of prolyl endopeptidase (EC 3.4.21.26), a cytosolic endopeptidase, which cleaves peptide bonds on the carboxylside of proline in proteins of relatively small molecular mass. Aims and methods: The aims of the present study were to examine serum PEP activity in patients with post-traumatic stress disorder (PTSD) versus healthy volunteers. PEP activity has been determined by a fluorimetric assay. Results: Serum PEP activity was significantly higher in patients with PTSD than in normal volunteers. Serum PEP activity was significantly higher in patients with PTSD and concurrent major depression than in patients with PTSD without major depression. In PTSD patients, there were no significant correlations between serum PEP activity and severity of PTSD symptoms. Conclusions: The results show that PTSD and, in particular, PTSD with concurrent major depression is associated with increased activity of PEP. Relevance: these results may be of importance for the (i) neuroendocrine pathophysiology of PTSD since PEP degrades neuropeptides, such as arginine vasopressin (AVP) and thyrotropin releasing hormone (TRH); and (ii) etiology of PTSD, since PEP degrades behaviorally active neuropeptides, such as AVP, TRH, oxytocin, neurotensin and substance P, which play a key role in positive reinforcement, social interactions, emotions and stress responsivity.

Lower Activity of Serum Peptidases in Abstinent Alcohol-Dependent Patients

This study examines i) the activity of serum prolyl endopeptidase (PEP) and dipeptidlyl peptidase IV (DPP IV) in detoxified alcohol-dependent patients without liver disease versus normal controls, and ii) the relationships between serum DPP IV and PEP activity and the production of cytokines or cytokine receptors, such as interleukin-6 (IL-6), tumor necrosis factor-α (TNF-α), interferon-γ (IFN-γ), IL-1 receptor antagonist (IL-1RA), and IL-10, and granulocyte-macrophage colony stimulatory factor (GM-CSF). Alcohol-dependent patients had significantly lower serum PEP and DPP IV activity than normal controls. We found that 58.3% and 50.0% of the alcohol-dependent patients, respectively, had PEP and DPP IV activities, which were lower than the mean control values minus 2 SD. There were significant inverse correlations between lowered serum DPP IV and PEP activity and the increased production of IL-6, INF-γ, IL-1RA, IL-10, and GM-CSF. These results show that lower serum DPP IV and PEP activity may be related to the pathophysiology of alcohol dependence.

Affected enzyme activities in Alzheimer's disease are sensitive to antemortem hypoxia.

Many enzyme activities in Alzheimer's disease (AD) are changed. Some of these enzyme activities are related to certain neurotransmitter systems. Enzymes in the brain can also be sensitive to antemortem hypoxia. In the present study it was determined if enzyme activities that are altered in AD are also subject to alteration by antemortem hypoxia. As an indicator of antemortem hypoxia brain lactate concentration was used. Enzyme activities measured were those of prolyl endopeptidase (PE), aminopeptidase (AP), phosphatidylinositol (PI) kinase, phosphatidylinositol phosphate kinase, alpha-ketoglutarate dehydrogenase (alpha-KGDH), choline acetyltransferase and beta-glucuronidase. All of these enzyme activities have been measured in AD patients before and several of them have been found to be decreased. In accordance with previous findings, PE, alpha-KGDH and ChAT activities were reduced in AD patients. PI kinase and beta-glucuronidase activities, however, were not reduced, contrary to previous findings. All enzyme activities, except that of beta-glucuronidase, correlated with brain lactate concentration, suggesting that antemortem hypoxia has a major influence on the activity of enzymes in the brain. PE, AP, alpha-KGDH and ChAT activities were still different between AD and control samples when these were matched for lactate concentration. The enzyme activities that were changed in AD were also significantly correlated with lactate concentration, an indicator of antemortem hypoxia, in brain specimens. This suggests that antemortem hypoxia and AD have some factor in common that may be responsible for changes in enzyme activities. Since both PE and alpha-KGDH are known to be sensitive to oxidative stress this factor could be oxidative stress.

EFFECTS OF PSYCHOLOGICAL STRESS ON SERUM PROLYL ENDOPEPTIDASE AND DIPEPTIDYL PEPTIDASE IV ACTIVITY IN HUMANS: HIGHER SERUM PROLYL ENDOPEPTIDASE ACTIVITY IS RELATED TO STRESS-INDUCED ANXIETY

There is now some evidence that psychiatric disorders, such as major depression, schizophrenia and post-traumatic stress disorder are associated with significant alterations in the serum activity of peptidases, such as prolyl endopeptidase (PEP) and dipeptidyl peptidase IV (DPP IV). The aims of the present study were to examine the effects of psychological stress on serum PEP and DPP IV activity in humans. Thirty-eight university students had repeated measurements of serum PEP and DPP IV activity a few weeks before and after (baseline conditions) as well as the day before a difficult academic examination (stress condition). Subjects were divided into anxiety responders and nonresponders to stress according to their stress-induced increase in the Spielberger State Anxiety Inventory. Serum PEP activity was somewhat lowered by stress in female, but not male, students. Serum PEP activity was significantly higher in the two baseline conditions and during the stress condition in anxiety responders than in anxiety nonresponders. There were no significant effects of stress on serum DPP IV activity and no significant differences between anxiety responders and nonresponders. Serum PEP and DPP IV activity were significantly higher in men than in women. The results suggest that increased baseline serum PEP activity is related to stress-induced anxiety.

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 expression 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.

Transient up-regulation of a prolyl endopeptidase activity in the microsomal fraction of rat liver during postnatal development.

To identify proteases which are involved in cell proliferation and differentiation of liver cells, we assayed various protease activities in rat liver during its postnatal development. We found that a protease activity specific to proline residues in the microsomal fraction of rat liver was transiently increased around postnatal day 8 and decreased thereafter, indicating that the enzyme activity is highly correlated to the proliferation and differentiation of liver cells. This protease was purified to an apparent homogeneity from the microsomal fraction of the postnatal-day-8 rat liver. The purified enzyme gave a single band with an apparent molecular mass of 65,000 on SDS/PAGE. It cleaved several peptide 4-methylcoumaryl-7-amide substrates and bioactive peptides at the C-terminus of proline residues. The enzyme did not hydrolyze any protein substrate examined suggesting that it is a peptidase rather than a proteinase. Although the best substrate among those tested was succinyl-Gly-Pro-Leu-Gly-Pro-4-methylcoumaryl-7-amide (-NH-Mec), the purified enzyme did not hydrolyze succinyl-Gly-Pro-NH-Mec, indicating that the enzyme has different substrate specificity from any hitherto known prolyl endopeptidases. These results suggest that the purified enzyme is a unique prolyl endopeptidase which may be involved in proliferation and differentiation of liver cells.

A serine protease from suspension-cultured soybean cells

A serine protease was purified from suspension-cultured soybean cells, by a combination of anion exchange, hydrophobic interaction and affinity chromatography. A 90000 M r subunit, which could be photoaffinity labelled with 3H-diisopropylfluorophosphate (DFP), was identified by SDS-polyacrylamide gel electrophoresis. The enzyme had a broad pH optimum from 5.5 to 8.5, and was strongly inhibited by antipain, leupeptin, aminoethylbenzenesulphonyl fluoride (AEBSF) and DFP, but not by soybean trypsin inhibitor. It cleaved several peptide 4-methylcoumaryl-7-amide derivatives after arginine or lysine residues. Mass spectroscopic analysis of oligopeptide digestion products indicated that the preferred cleavage positions were between paired arginine residues, or C-terminal to single arginine residues, depending on the oligopeptide substrate. Partial amino acid sequences from the purified protein showed sequence identity to bacterial protease II and prolyl peptidase, although the enzyme lacked prolyl endopeptidase activity. We discuss the possible involvement of the protease in plant defense responses.

A novel prolyl endopeptidase inhibitor, JTP-4819--its behavioral and neurochemical properties for the treatment of Alzheimer's disease.

Formation of beta-amyloid and neurofibrillary tangles in the brain due to genetic or other factors is the most frequent cause of Alzheimer's disease. In addition, marked reduction of certain brain neuropeptide levels is a consistent finding in patients with Alzheimer's disease, together with the deterioration of cholinergic neurons. Currently, there is great demand for the development of new drugs to improve memory deficits or to delay the neurodegenerative process in conditions such as Alzheimer's disease. In this report, the pharmacological actions of JTP-4819, a novel specific prolyl endopeptidase (PEP) inhibitor devised for the treatment of Alzheimer's disease, are reviewed with respect to its effects on PEP activity, neuropeptidergic and cholinergic neurons, and memory-related behavior in rats. We also discuss the possible beneficial effect of JTP-4819 on beta-amyloid metabolism and its potential neuroprotective properties.

Peptidases that degrade gonadotropin-releasing hormone: influence on LH secretion in the ewe.

Several peptidases have been postulated to degrade the hypothalamic peptide gonadotropin-releasing hormone (GnRH), but it is not known if such enzymes contribute significantly to the delivery of GnRH to the pituitary in vivo. Furthermore, the activity of GnRH-inactivating peptidases may vary in different reproductive states, such as across the estrous cycle. In the present study, specific fluorescent substrates were used to measure the activity of the two major GnRH-degrading enzymes, prolyl endopeptidase (PEP) and endopeptidase 3.4.24.15 (EP 24.15), in soluble extracts of the median eminentes (ME) of ewes during different phases of the estrous cycle. Levels of EP 24.15 and PEP activity in the ME did not vary significantly across the cycle, although PEP activity was lowest at the time of the preovulatory luteinizing hormone (LH) surge. However, a statistically significant decline in PEP activity (18%, P = 0.02) was observed in the ME of OVX ewes in which a surge was induced by estrogen when compared to oil-treated OVX controls, suggesting a possible negative regulation of PEP activity by this steroid. The effect of intracerebroventricular (i.c.v.) infusion of several peptidase inhibitors on the pulsatile release of LH in the conscious OVX ewe was also examined. No consistent changes in the pattern of LH release were observed with i.c.v. infusion of the EP 24.15 inhibitor N-[1(R,S)-carboxy-3-phenylpropyl]-Ala-Ala-Tyr-p-aminobenzoate (cFP-AAY-pAB) or the angiotensin-converting enzyme (ACE) inhibitor captopril. Similarly, administration of the prolyl endopeptidase inhibitor bacitracin, or a more specific inhibitor of this enzyme, Z-Proprolinal (ZPP), did not alter LH release patterns. The results did not demonstrate a major role for changes in the activity of EP 24.15, PEP, or ACE in altering the pattern of GnRH secretion, but a minor reduction in PEP levels may occur at the time of the estrogen-induced LH surge.

A novel prolyl endopeptidase inhibitor, JTP-4819, with potential for treating Alzheimer's disease

The pharmacological actions of JTP-4819, a new prolyl endopeptidase (PEP) inhibitor targeted for the treatment of Alzheimer's disease, are reviewed with respect to its effects on PEP activity, brain neurotransmitters, and memory-related behaviour in rats. JTP-4819 was shown to be a very potent and specific inhibitor of PEP. At nanomolar concentration, JTP-4819 inhibited the degradation of substance P, arginine-vasopressin, and thyrotropin-releasing hormone by PEP in supernatants of the rat cerebral cortex and hippocampus. Repeated administration of JTP-4819 reversed the aging-induced decrease in brain substance P-like and thyrotropin-releasing hormone-like immunoreactivity, suggesting that this drug may be able to improve the imbalance of peptidergic neuronal systems that develops with senescense by inhibiting PEP activity. JTP-4819 increased acetylcholine release from the frontal cortex and hippocampus, regions closely associated with memory, in both young and aged rats. In addition, it improved performance in several memory and learning-related tests (e.g., the Morris water maze task in aged or MCA-occluded rats and the passive avoidance test). This memory-enhancing effect of JTP-4819 may result from prevention of the metabolic degradation of brain neuropeptides by PEP as well as from the enhancement of acetylcholine release. Taken together, these unique and potent pharmacological actions of JTP-4819 suggest that it may have the potential to be used for treating Alzheimer's disease.

Morphine withdrawal alters anterior pituitary hormone secretion, brain endopeptidase activity and brain monoamine metabolism in the rat.

Rats were made tolerant to morphine by a 5-day regimen with increasing doses. The time course of changes in serum anterior pituitary hormone levels, brain endo- and exopeptidase activity, levels of brain biogenic amines and body weight were studied during abrupt morphine withdrawal. Cold stimulated secretion of thyrotropin and the secretion of growth hormone were both decreased whereas that of prolactin was increased. In the hypothalamus both prolyl endopeptidase and dipeptidyl peptidase IV activities were concomitantly increased. The hypothalamic 5 hydroxyindole acetic acid levels were also increased. Changes in hormone secretion, peptidase activity and monoamine turnover had returned to baseline levels by 92 hr. Our results indicate that morphine withdrawal and the associated stress produce alterations in anterior pituitary thyrotropin and growth hormone secretion. Concomitant increases in hypothalamic prolyl endopeptidase and dipeptidyl peptidase activities may contribute to these changes.

Effect of a novel prolyl endopeptidase inhibitor, JTP-4819, on neuropeptide metabolism in the rat brain

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.

Serum Prolyl Endopeptidase Activities of Patients with Senile Dementia of the Alzheimer Type and of Those with Vascular Dementia.

The serum prolyl endopeptidase activities of patients with senile dementia of the Alzheimer type (SDAT) and of those with vascular dementia (VD) were measured by a sensitive fluorometric assay. As a result, the prolyl endopeptidase activity of both SDAT patients (p<0.025) and VD patients (p<0.001) was found to be significantly lower than that of normal subjects, and this activity of VD patients turned out to be significantly lower (p<0.001) than that of SDAT patients. The correlation between serum prolyl endopeptidase activity and that of other serum enzymes was examined, and a significant correlation was found with creatine kinase activity (r=0.560, p<0.001). Judging from these observations, it seems possible that the brain disorders of the dementia patients may have affected the metabolism of skeletal muscles, which possibly resulted in the lowering of their prolyl endopeptidase activity. At present, there are very few markers available for physiological diagnosis to distinguish between SDAT and VD patients, and therefore, the measurement of serum prolyl endopeptidase activities of dementia patients seems to be beneficial.

Alterations in plasma prolyl endopeptidase activity in depression, mania, and schizophrenia: effects of antidepressants, mood stabilizers, and antipsychotic drugs

The activity of prolyl endopeptidase (PEP), a serine proteinase, has been found to be significantly lower in the blood of patients with major depression than in normal volunteers. The present study investigates plasma PEP activity in 25 major depressed, 10 manic, and 14 schizophrenic subjects versus 30 normal volunteers. It also examines the effects of antidepressants, valproate, and neuroleptic drugs on plasma PEP activity. PEP activity was significantly lower in major depressed subjects than in normal volunteers and in patients with mania and schizophrenia. In depressed subjects, plasma PEP activity was significantly increased during treatment with antidepressant drugs, such as fluoxetine. Plasma PEP activity was significantly increased in manic and schizophrenic subjects compared with normal volunteers. In manic subjects, short-term treatment with valproate had a significant suppressive effect on PEP activity. No significant effects of neuroleptics on PEP activity could be found in the schizophrenic patients. The results support the hypothesis that lower PEP activity could play a role in the pathophysiology of major depression, while increased PEP activity may be related to psychotic conditions, such as mania and schizophrenia.

Effect of a novel prolyl endopeptidase inhibitor, JTP-4819, on prolyl endopeptidase activity and substance P- and arginine-vasopressin-like immunoreactivity in the brains of aged rats.

The effects of a novel prolyl endopeptidase (PEP) inhibitor, (S)-2-[[(S)-2-(hydroxyacetyl)-1-pyrrolidinyl]carbonyl]-N-(phenylmethyl) - 1-pyrrolidinecarboxamide (JTP-4819), on the PEP activity in the brain and on the contents of substance P (SP)- and arginine-vasopressin (AVP)-like immunoreactivity (LI) in the cerebral cortex and hippocampus of young and aged rats were investigated using enzyme immunoassay. JTP-4819 exhibited a concentration-dependent in vitro inhibitory action on PEP activity in the brains of both young and aged rats, with IC50 values of approximately 0.7 and 0.8 nM, respectively. A single dose of JTP-4819 (3 mg/kg, p.o.) increased the SPLI content in the cerebral cortex but not the hippocampus of aged rats (23-24 months old). In addition, repeated administration of JTP-4819 (1 mg/kg, p.o., for 21 days) increased the SPLI content in the cerebral cortex and restored the SPLI content in the hippocampus, which had decreased with aging. In contrast, single (1 mg/kg, p.o.) and repeated (1 mg/kg, p.o., for 21 days) administration of JTP-4819 only tended to increase the AVPLI content of the hippocampus and cerebral cortex in aged rats, respectively. These results indicate that JTP-4819 increases the cerebral and hippocampal SPLI content in aged rats by inhibiting the action of PEP.

Identification and localisation of a synaptosomal membrane prolyl endopeptidase from bovine brain.

Prolyl endopeptidase, which has long been recognised for its importance in the degradation of several neuropeptides such as thyroliberin, luteinising hormone releasing hormone, angiotensin, substance P and neurotensin, has been widely characterised as a cytosolic enzyme. However, in this paper, we report the presence of a prolyl endopeptidase activity in the particulate fractions of bovine brain, which is distinct from that in the cytoplasm. This previously uncharacterised activity was found to reside in the synaptosomal membranes, a location which is highly significant for the inactivation of neuropeptides in brain. Following vigorous salt washing and osmotic shock, the prolyl endopeptidase activity was released from the membranes by treatment with the detergent Triton X-100, and was partially purified by gel filtration on a Sephacryl S-200HR column. This prolyl endopeptidase activity was shown to have a molecular mass (87 kDa) higher than the cytosolic prolyl endopeptidase but, from initial investigation, appears to demonstrate a similarly broad substrate specificity towards proline-containing neuropeptides. The partially purified enzyme was inhibited by certain thiol-protease inhibitors and was also found to be sensitive to the metal chelator 1,10-phenanthroline.

A sensitive detection method for peptide using 4-fluoro-7-nitrobenzo-2-oxa-1,3-diazole and its application to measure prolyl endopeptidase activity.

A measuring method sensitive to prolyl endopeptidase (EC 3.4.21.26, PEP) activity using native peptides (Arg-vasopressin or substance P) as substrates was established. The investigation of three different derivatization reagents, which had been developed for an amino acid analysis, demonstrated that 4-fluoro-7-nitrobenzo-2-oxa-1,3-diazole (NBDF) was the most suitable for the detection of Arg-Gly-NH2, which was released from Arg-vasopressin by PEP. Arg-Gly-NH2 was reacted with NBDF at 65 degrees C for 5 min at pH 7.6 and the reaction mixture was analysed by HPLC on a reverse-phase column by monitoring the fluorescence intensity. The detection limit was 1 picomol per injection and the linear standard calibration curve could be constructed in the range of 1 to 100 picomol per injection with a 3.0% relative standard deviation. This sensitive detection method for peptide was applied to the measurement of PEP activity using Arg-vasopressin as a substrate and 1 x 10(-3) unit of PEP activity was detectable. This method was also applicable to the measurement of PEP activity using substance P as a substrate by detecting the derivative of its fragment peptide (Arg-Pro-Lys-Pro).

Age-related change of prolyl endopeptidase activity in rat brain

Age-related change of prolyl endopeptidase activity in rat brain

Evidence for a two-step mechanism of gonadotropin-releasing hormone metabolism by prolyl endopeptidase and metalloendopeptidase EC 3.4.24.15 in ovine hypothalamic extracts.

The metalloendopeptidase EC 3.4.24.15 is believed to degrade gonadotropin-releasing hormone (GnRH) (pGlu-His-Trp-Ser-Tyr-Gly-Leu-Arg-Pro-Gly-NH2) by cleavage at the Tyr5-Gly6 bond. We compared the ability of crude and partially purified endopeptidase 24.15 from ovine hypothalamus with recombinant rat testicular endopeptidase 24.15 to degrade synthetic GnRH. Both soluble and membrane hypothalamic fractions degraded GnRH to GnRH1-5, with some production of GnRH1-9 and GnRH1-3. Generation of the smaller fragments was blocked by a specific endopeptidase 24.15 inhibitor (CFP-AAY-pAB), but production of GnRH1-9 was reciprocally enhanced, suggesting this peptide may be an intermediate generated by prolyl endopeptidase. Indeed, both bacitracin and Z-Pro-prolinal, inhibitors of this enzyme, markedly reduced GnRH degradation to any product. Degradation of synthetic GnRH1-9 was more rapid than that of GnRH and was inhibited by CFP-AAY-pAB but not bacitracin. Activity against either substrate was greater in the soluble fraction. Repeated washing of the membrane fraction followed by extraction with Triton X-114 suggested that both endopeptidase 24.15 and prolyl endopeptidase, although predominantly soluble, can be peripherally associated with membranes. When fractionated by hydrophobic interaction chromatography, soluble endopeptidase 24.15 degraded GnRH only in fractions that also exhibited prolyl endopeptidase activity. In contrast, maximal degradation of GnRH1-9 was observed in adjacent fractions, which also contained the highest levels of immunoreactive endopeptidase 24.15. The affinity of recombinant endopeptidase 24.15 for GnRH was low (Km = 1.35 mM), was improved 10-15-fold by removal of the COOH-terminal amide or glycinamide (Km = 90 and 119 microM, respectively), and could be inhibited by CFP-AAY-pAB but not bacitracin. Taken together, these results suggest that GnRH metabolism in the hypothalamus may occur via a two-step process involving first removal of Gly10-NH2 by prolyl endopeptidase, followed by cleavage by endopeptidase 24.15 at the Tyr5-Gly6 bond.

Molecular cloning and characterization of prolyl endopeptidase from human T cells.

The prolyl endopeptidase (PEP) gene of human T cells was amplified by the PCR method and cloned in Escherichia coli. The complete gene consisted of 2,130 nucleotides corresponding to 710 amino acid residues with a calculated molecular mass of 80,750. The nucleotide sequence of this clone revealed that T cell PEP DNA is 48, 50, and 91% homologous to those of Flavobacterium meningosepticum, Aeromonas hydrophila, and porcine brain PEP, respectively. This gene was fused to the lacZ sequence from E. coli and expressed as a fused protein in E. coli. This fused protein exhibited PEP activity, which was inhibited by Z-Pro-prolinal, a specific inhibitor of PEP. The fused protein was purified on a beta-galactosidase specific affinity column. A polyclonal antibody was raised against the purified protein. Immunological characterization suggested that this protein is different from cytosol-soluble PEP.