Kidney neutral endopeptidase and the hydrolysis of enkephalin by synaptic membranes show similar sensitivity to inhibitors.
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ABSTRACT:
SUBMITTER: Fulcher IS
PROVIDER: S-EPMC1158259 | biostudies-other | 1982 May
REPOSITORIES: biostudies-other
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Project description:Melanin-concentrating hormone (MCH) is a cyclic peptide which behaves as an antagonist of the pituitary melanotropic hormone alpha-melanocyte-stimulating hormone in fishes. Cloning of the rat MCH cDNA precursor recently revealed the presence of an additional putative peptide named NEI. The present work examined the susceptibility of these novel peptides to hydrolysis by various purified exo- and endo-peptidases including endopeptidases 24.11 (NEP), 24.15, 24.16, angiotensin-converting enzyme, leucine aminopeptidase and carboxypeptidase A. NEP attacked MCH at three sites of the molecule with an apparent affinity of about 12 microM and a kcat. of 4 min-1. The first site of cleavage was at Cys-7-Met-8, i.e. within the peptide loop formed by the internal disulphide bridge. NEP could therefore be considered as an MCH-inactivating peptidase since the degradation products generated are probably devoid of biological activity. In contrast, NEI neither inhibited the degradation of the NEP chromogenic substrate glutaryl-Phe-Ala-Phe-p-aminobenzoate nor was susceptible to proteolysis by NEP. Unlike NEP, angiotensin-converting enzyme, endopeptidase 24.15 and endopeptidase 24.16 appeared totally unable to cleave MCH, whereas the peptide was readily degraded by aminopeptidase M and carboxypeptidase A.
| S-EPMC1133042 | biostudies-other
Project description:We investigated the precision of the approximate number system (ANS) in three lemur species (Lemur catta, Eulemur mongoz, and Eulemur macaco flavifrons), one Old World monkey species (Macaca mulatta) and humans (Homo sapiens). In Experiment 1, four individuals of each nonhuman primate species were trained to select the numerically larger of two visual arrays on a touchscreen. We estimated numerical acuity by modeling Weber fractions (w) and found quantitatively equivalent performance among all four nonhuman primate species. In Experiment 2, we tested adult humans in a similar procedure, and they outperformed the four nonhuman species but showed qualitatively similar performance. These results indicate that the ANS is conserved over the primate order.
| S-EPMC3966981 | biostudies-literature
Project description:Racecadotril (acetorphan) is a neutral endopeptidase (NEP) inhibitor with known antidiarrheal activity in animals and humans; however, in humans, it suffers from shortcomings that might be improved with newer drugs in this class that have progressed to the clinic for nonenteric disease indications. To identify potentially superior NEP inhibitors with immediate clinical utility for diarrhea treatment, we compared their efficacy and pharmacologic properties in a rat intestinal hypersecretion model. Racecadotril and seven other clinical-stage inhibitors of NEP were obtained or synthesized. Enzyme potency and specificity were compared using purified peptidases. Compounds were orally administered to rats before administration of castor oil to induce diarrhea. Stool weight was recorded over 4 hours. To assess other pharmacologic properties, select compounds were orally administered to normal or castor oil-treated rats, blood and tissue samples collected at multiple time points, and active compound concentrations determined by mass spectroscopy. NEP enzyme activity was measured in tissue homogenates. Three previously untested clinical NEP inhibitors delayed diarrhea onset and reduced total stool output, with little or no effect on intestinal motility assessed by the charcoal meal test. Each was shown to be a potent, highly specific inhibitor of NEP. Each exhibited greater suppression of NEP activity in intestinal and nonintestinal tissues than did racecadotril and sustained this inhibition longer. These results suggest that newer clinical-stage NEP inhibitors originally developed for other indications may be directly repositioned for treatment of acute secretory diarrhea and offer advantages over racecadotril, such as less frequent dosing and potentially improved efficacy.
| S-EPMC4851326 | biostudies-literature
Project description:A search for the natural substrates for neutral endopeptidase (NEP; EC 3.4.24.11) in the immune system led to investigation of the enzyme's action on thymic humoral factor gamma 2 (THF). The ectoenzyme rapidly and efficiently hydrolyses the Lys6-Phe7 bond of the octapeptide. The site of cleavage was confirmed by h.p.l.c. analysis, amino acid analysis and sequence determination of the products. Phosphoramidon (3.6 microM), a potent inhibitor of the enzyme, prevents this cleavage even during prolonged incubation. The high efficiency of hydrolysis of THF by NEP is similar to that reported for [Leu5]enkephalin, and the dipeptide Phe-Leu is the C-terminal product in the hydrolysis of both peptides. The presence of NEP, reportedly identified as the common acute lymphoblastic leukaemia antigen (CALLA), in bone-marrow cells and other cells of the immune system raises the possibility that it may play a role in modulating the activity of peptides such as THF.
| S-EPMC1151431 | biostudies-other
Project description:Locust adipokinetic hormone (AKH, pGlu-Leu-Asn-Phe-Thr-Pro-Asn-Trp-Gly-Thr-NH2) was used as the substrate to measure neuropeptide-degrading endopeptidase activity in neutral membranes from ganglia of the locust Schistocerca gregaria. Initial hydrolysis of AKH at neural pH by peptidases of washed neural membranes generated pGlu-Leu-Asn and Phe-Thr-Pro-Asn-Trp-Gly-Thr-NH2 as primary metabolites, demonstrating that degradation was initiated by cleavage of the Asn-Phe bond. Amastatin protected the C-terminal fragment from further metabolism by aminopeptidase activity without inhibiting AKH degradation. The same fragments were generated on incubation of AKH with purified pig kidney endopeptidase 24.11, and enzyme known to cleave peptide bonds that involve the amino group of hydrophobic amino acids. Phosphoramidon (10 microM), a selective inhibitor of mammalian endopeptidase 24.11, partially inhibited the endopeptidase activity of locust neural membranes. This phosphoramidon-sensitive activity was shown to enriched in a synaptic membrane preparation with around 80% of the activity being inhibited by 10 microM-phosphoramidon (IC50 = 0.2 microM). The synaptic endopeptidase was also inhibited by 1 mM-EDTA, 1 mM-1,10-phenanthroline and 1 microM-thiorphan, and the activity was maximal between pH 7.3 and 8.0. Localization of the phosphoramidon-sensitive enzyme in synaptic membranes is consistent with a physiological role for this endopeptidase in the metabolism of insect peptides at the synapse.
| S-EPMC1135318 | biostudies-other
Project description:To protect against HIV infection, passively transferred and/or vaccine-elicited neutralizing antibodies (NAbs) need to effectively target diverse subtypes that are transmitted globally. These variants are a limited subset of those present during chronic infection and display some unique features. In the case of mother-to-child transmission (MTCT), transmitted variants tend to be resistant to neutralization by maternal autologous NAbs.To investigate whether variants transmitted during MTCT are generally resistant to HIV-1-specific NAbs, 107 maternal or infant variants representing the dominant HIV-1 subtypes were tested against six recently identified HIV-1-specific broadly neutralizing monoclonal antibodies (bNAbs), NIH45-46W, VRC01, PGT128, PGT121, PG9 and PGT145.Infant and maternal variants did not differ in their neutralization sensitivity to individual bNAbs, nor did viruses from transmitting versus nontransmitting mothers, although there was a trend for viruses from transmitting mothers to be less sensitive overall. No single bNAb neutralized all viruses, but a combination of bNAbs that target distinct epitopes covered 100% of the variants tested. Compared with heterosexually transmitted variants, vertically transmitted variants were significantly more sensitive to neutralization by PGT128 and PGT121 (P=0.03 in both cases), but there were no differences for the other bNAbs. Overall, subtype A variants were significantly more sensitive to NIH45-46 (P=0.04), VRC01 (P=0.002) and PGT145 (P=0.03) compared with the nonsubtype A and less sensitive to PGT121 than subtype Cs (P=0.0001).A combination of bNAbs against distinct epitopes may be needed to provide maximum coverage against viruses in different modes of transmission and diverse subtypes.
| S-EPMC4080909 | biostudies-literature
Project description:Mycophenolic acid (MPA) appears to have anti-fibrotic effects, but the molecular mechanisms underlying this are unknown. We prospectively studied 35 stable kidney transplant recipients maintained on cyclosporine and azathioprine. We converted 20 patients from azathioprine to enteric-coated mycophenolate sodium (EC-MPS) and continued the remaining 15 patients on azathioprine. Exploratory mRNA expression profiling, performed on five randomly selected EC-MPS patients, revealed significant upregulation of neutral endopeptidase (NEP), which is an enzyme that degrades angiotensin II. We confirmed these microarray data by measuring levels of NEP expression in all subjects; in addition, we found that NEP gene expression correlated inversely with proteinuria. In an additional 33 patients, glomerular and tubular NEP protein levels from renal graft biopsies were significantly higher among the 13 patients receiving cyclosporine + EC-MPS than among the 12 patients receiving cyclosporine + azathioprine or 8 patients receiving cyclosporine alone. Glomerular NEP expression inversely correlated with glomerulosclerosis and proteinuria, and tubular NEP expression inversely correlated with interstitial fibrosis. Incubation of human proximal tubular cells with MPA increased NEP gene expression in a dose- and time-dependent manner. Moreover, MPA reduced angiotensin II-induced expression of the profibrotic factor plasminogen activator inhibitor-1, and a specific NEP inhibitor completely reversed this effect. Taken together, our data suggest that MPA directly induces expression of neutral endopeptidase, which may reduce proteinuria and slow the progression of renal damage in kidney transplant recipients.
| S-EPMC3014029 | biostudies-literature
Project description:Neutral endopeptidase-24.11 (EC 3.4.24.11) (NEP) is a transmembrane metallo-endopeptidase that has been shown to be involved in the degradation of several mammalian neuropeptides, including enkephalins. The enzyme has recently been found to be specifically associated with the axonal and synaptic membranes of neurons in the globus pallidus of the pig brain. This result suggests that neurons must possess mechanisms for targeting NEP to particular membrane domains. Study of these mechanisms would greatly benefit from the existence of an established neuron-like cell line capable of expressing and targeting NEP to specific membrane domains. For this reason we have used a retroviral vector containing the cDNA for rabbit kidney NEP to express this enzyme in a mouse neuroblastoma cell line (Neuro2A). Labelling of the cell surface with an antibody coupled to colloidal gold particles and examination of the cells by electron microscopy revealed a non-uniform distribution of NEP at the surface of the cells, the protein being preferentially associated with the membrane of neurites compared with the cell body. This observation suggests that Neuro2A cells possess a mechanism for targeting NEP to specific domains of the plasma membrane. This cell line could thus constitute a good model for studying the mechanisms responsible for targeting this enzyme to specialized regions of the plasma membrane.
| S-EPMC1131309 | biostudies-other
Project description:Neutral endopeptidase (NEP) is a 90- to 110-kDa cell-surface peptidase that is normally expressed by numerous tissues but whose expression is lost or reduced in a variety of malignancies. The anti-tumorigenic function of NEP is mediated not only by its catalytic activity but also through direct protein-protein interactions of its cytosolic region with several binding partners, including Lyn kinase, PTEN, and ezrin/radixin/moesin (ERM) proteins. We have previously shown that mutation of the K(19)K(20)K(21) basic cluster in NEPs' cytosolic region to residues QNI disrupts binding to the ERM proteins. Here we show that the ERM-related protein merlin (NF2) does not bind NEP or its cytosolic region. Using experimental data, threading, and sequence analysis, we predicted the involvement of moesin residues E(159)Q(160) in binding to the NEP cytosolic domain. Mutation of these residues to NL (to mimic the corresponding N(159)L(160) residues in the nonbinder merlin) disrupted moesin binding to NEP. Mutation of residues N(159)L(160)Y(161)K(162)M(163) in merlin to the corresponding moesin residues resulted in NEP binding to merlin. This engineered NEP peptide-merlin interaction was diminished by the QNI mutation in NEP, supporting the role of the NEP basic cluster in binding. We thus identified the region of interaction between NEP and moesin, and engineered merlin into a NEP-binding protein. These data form the basis for further exploration of the details of NEP-ERM binding and function.
| S-EPMC2771306 | biostudies-literature
Project description:Cell surface metallo-endopeptidases play important roles in cell communication by controlling the levels of bioactive peptides around peptide receptors. To understand the relative relevance of these enzymes in the CNS, we characterized a metallo-endopeptidase in the CNS of Aplysia californica, whose peptidergic pathways are well described at the molecular, cellular, and physiological levels. The membrane-bound activity cleaved Leu-enkephalin at the Gly3-Phe4 bond with an inhibitor profile similar to that of the mammalian neutral endopeptidase (NEP). This functional homology was supported by the molecular cloning of cDNAs from the CNS, which demonstrated that the Aplysia and mammalian NEPs share all the same amino acids that are essential for the enzymatic activity. The protein is recognized both by specific anti-Aplysia NEP (apNEP) antibodies and by the [125I]-labeled NEP-specific inhibitor RB104, demonstrating that the apNEP gene codes for the RB104-binding protein. In situ hybridization experiments on sections of the ganglia of the CNS revealed that apNEP is expressed in neurons and that the mRNA is present both in the cell bodies and in neurites that travel along the neuropil and peripheral nerves. When incubated in the presence of a specific NEP inhibitor, many neurons of the buccal ganglion showed a greatly prolonged physiological response to stimulation, suggesting that NEP-like metallo-endopeptidases may play a critical role in the regulation of the feeding behavior in Aplysia. One of the putative targets of apNEP in this behavior is the small cardioactive peptide, as suggested by RP-HPLC experiments. More generally, the presence of apNEP in the CNS and periphery may indicate that it could play a major role in the modulation of synaptic transmission in Aplysia and in the metabolism of neuropeptides close to their point of release.
| S-EPMC6782589 | biostudies-literature