Gene/Protein Disease Symptom Drug Enzyme Compound
Pivot Concepts:   Target Concepts:
Query: EC:3.4.24.11 (CD10)
 9,792 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

The human colon cancer cell line Caco-2 undergoes spontaneous enterocytic differentiation during growth, and expresses a number of brush-border-membrane-associated hydrolases typical of a differentiated phenotype. Among these are alkaline phosphatase, dipeptidyl peptidase IV and sucrase-isomaltase (sucrase, EC 3.2.1.48). Neutral endopeptidase 24.11 [EC 3.4.24.11, neprilysin (NEP)] is another abundant protease of normal enterocytes but its presence in Caco-2 cells has not been fully documented yet. In this paper, we show that Caco-2 cell extracts hydrolyse tritiated [D-Ala2Leu5]enkephalin with a Km of 180 microM, very close to the value obtained for the NEP present in the rabbit kidney (118 microM). Western-blot analysis of brush-border membranes purified from post-confluent cells revealed a protein with an apparent molecular mass of 94000 Da similar to that of the rabbit kidney NEP. The amount of enzyme in cell extracts increased as a function of the age of the culture, indicating that NEP expression is correlated with the degree of cell differentiation as is also the case for sucrase and dipeptidylpeptidase IV (DPP-IV). Binding of a radiolabelled antibody to Caco-2 cell monolayers grown on semi-permeable filters indicated that 95% of NEP molecules present at the cell surface are on the apical side. Immunocytochemical and flow cytometric analysis of intact and permeabilized cells were also used to investigate the presence of NEP and DPP-IV at the surface of Caco-2 cells. Whereas DPP-IV staining appeared to be homogeneous throughout the entire cell population, NEP-related fluorescence exhibited a bimodal distribution which indicates an uneven expression of the protein at the cell surface. Permeabilization of monolayers with saponin before staining restored a labelling pattern for NEP similar to the one obtained for DPP-IV. This suggests that although DPP-IV and NEP follow similar patterns of expression when enzymic activities are measured on whole-cell extracts, targeting of these brush-border proteins to the cell surface appears to be regulated in different ways.
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PMID:Polarized distribution of neutral endopeptidase 24.11 at the cell surface of cultured human intestinal epithelial Caco-2 cells. 136 26

In vivo, bicarbonate can affect proximal tubule intermediary metabolism, including gluconeogenesis, ammoniagenesis and maintenance of the mitochondrial substrate supply. In vitro, rabbit proximal tubule cells (RPTC) in primary culture revert from gluconeogenesis to glycolysis and their mitochondrial metabolism remains lower than in vivo. To determine whether the bicarbonate buffer system could have an effect on these deregulations, RPTC in primary culture grown in the absence of insulin and glucose in the culture medium were developed either with the standard sodium bicarbonate buffer with 5% CO2 or with a Hepes hydrogen ion buffer in the presence of 0.5% CO2. Duration of the bicarbonate-free cultures was increased until at least day 17 after seeding, compared with day 11 in bicarbonate-buffered cultures. As could be expected, succinate dehydrogenase activity remained stable as a function of time in bicarbonate-free cultures while an early marked decrease of this activity occurred from seeding in cultures developed in the presence of bicarbonate buffer. Compared to bicarbonate-buffered cells, higher phosphoenolpyruvate carboxykinase activity concomitant with lower intracellular lactate dehydrogenase activity was observed in cultures developed in the absence of bicarbonate, which is indicative of closer carbohydrate metabolism orientation to the in vivo situation for RPTC. Immunofluorescence staining of RPTC with monoclonal antibodies directed to neutral endopeptidase (NEP), and dipeptidyl-peptidase IV (DPP II) showed similar extensive labelling with DPP and NEP in both culture conditions. Confocal microscopy analysis of NEP subcellular distribution, showed exclusive targetting of NEP to the apical plasma membranes. In both models, cAMP production was stimulated by parathyroid hormone and unaffected by arginine vasopressin. In conclusion, bicarbonate withdrawal from the culture medium (without changing the pH of the medium) allows a marked improvement of mitochondrial capacity and carbohydrate metabolism pattern without any loss of differentiated properties.
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PMID:Effects of the medium HCO3-/CO2 buffer system on differentiation and intermediary metabolism properties of rabbit proximal tubule cells in primary culture. 897 88

A novel dipeptidylpeptidase (DPP-7) was purified from the membrane fraction of Porphyromonas gingivalis. This enzyme, with an apparent molecular mass of 76 kDa, has the specificity for both aliphatic and aromatic residues in the P1 position. Although it belongs to the serine class of peptidases, it does not resemble other known dipeptidylpeptidases. Interestingly, the amino acid sequence around the putative active site serine residue shows significant similarity to the C-terminal region of the Staphylococcus aureus V-8 endopeptidase. The genes encoding homologues of DPP-7 were found in genomes of Xylella fastidiosa, Shewanella putrefaciens, and P. gingivalis. It is likely that at least in P. gingivalis, DPP-7 and its homologue, in concert with other di- and tripeptidases, serve nutritional functions by providing dipeptides to this asaccharolytic bacterium.
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PMID:Porphyromonas gingivalis DPP-7 represents a novel type of dipeptidylpeptidase. 1109 98

The Ministry of Education, Culture and Social Welfare (MOECSW), as part of the Population Education Programs (formal and informal), undertook a series of training programs to upgrade the knowledge and skills of master trainers, supervisors, and resource persons. As part of the Population Education in the Formal School Sector Project (NEP/93/P01), under the Curriculum Development Centre five training courses were organized to train 220 master trainers. Under the "Three Steps Training Strategy," these 220 master trainers would teach 825 secondary school headmasters who would reach 2025 secondary school teachers. The training courses were held in Dhangadi, April 23-27, 1995; in Pokhara, April 2-7; and in Biratnagar, February 20-24. The areas covered included: 1) the pedagogical aspect of population education (content, scope, objectives, nature, teaching methodologies); 2) demography and population dynamics (composition, distribution and density, sources of population data, demographic transition, consequences and determinants of population growth); 3) family life and adolescence and human sexuality education, including acquired immunodeficiency syndrome (AIDS) education; 4) maternal and child health, and family planning; 5) environment; and 6) population policy and programs. As part of the Population Education Programme (NEP/93/P08), a Master Trainers Training Workshop was held in Makwanpur, March 26-28, 1995. These master trainers would train trainers who would train the facilitators and teachers at learning centers for adult learners under the literacy and post literacy programs. This course focused on the approaches and strategies for integrating population education in development programs, and non-formal education, adult literacy, post literacy, and out-of-school children programs. Dr. D. de Rebello and Mr. S. Hutabarat, CST Advisors on Population Education, organized the training courses and served as resource persons.
Popul Educ Asia Pac Newsl Forum 1995
PMID:MOECSW trains master trainers and supervisors. 1231 59

Small membranous vesicles, between 25- and 75-nm diameter, were collected by high-speed centrifugation from the ram cauda epididymal fluid and were found to be normal constituents of this fluid and of the seminal plasma. The SDS-PAGE protein pattern of these vesicles was specific and very different from that of the caudal fluid, seminal plasma, sperm extract, and cytoplasmic droplets. After two-dimensional electrophoresis separation and mass spectrometry analysis, several proteins were identified and grouped into i) membrane-linked enzymes, such as dipeptidyl peptidase IV (DPP-IV), neprilysin (NEP), phosphodiesterase-I (E-NPP3), and protein G-beta; ii) vesicle-associated proteins, such as lactadherin (MFEG8-PAS6/7) and vacuolar ATPase; iii) several cytoskeleton-associated proteins, such as actin, ezrin and annexin; and iv) metabolic enzymes. The presence of some of these proteins as well as several different hydrophobic proteins secreted by the epididymis was further confirmed by immunoblotting. These markers showed that the majority of the vesicles originated from the cauda epididymal region. The physical and biochemical characteristics of these vesicles suggest they are the equivalent of the exosomes secreted by several cell types and epithelium. The main membrane-linked proteins of the vesicles were not retrieved in the extract from cauda or ejaculated sperm, suggesting that these vesicles did not fuse with sperm in vivo.
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PMID:Identification, proteomic profiling, and origin of ram epididymal fluid exosome-like vesicles. 1563 28

Fibroblast activation protein (FAP) and dipeptidyl peptidase-4 (DPP-4) are highly homologous serine proteases of the prolyl peptidase family and therapeutic targets for cancer and diabetes, respectively. Both proteases display dipeptidyl peptidase activity, but FAP alone has endopeptidase activity. FAP Ala657, which corresponds to DPP-4 Asp663, is important for endopeptidase activity; however, its specific role remains unclear, and it is unknown whether conserved DPP-4 substrate binding residues support FAP endopeptidase activity. Using site-directed mutagenesis and kinetic analyses, we show here that Ala657 and five conserved active site residues (Arg123, Glu203, Glu204, Tyr656, and Asn704) promote FAP endopeptidase activity via distinct mechanisms of transition state stabilization (TSS). The conserved residues provide marked TSS energy for both endopeptidase and dipeptidyl peptidase substrates, and structural modeling supports their function in binding both substrates. Ala657 also stabilizes endopeptidase substrate binding and additionally dictates FAP reactivity with transition state inhibitors, allowing tight interaction with tetrahedral intermediate analogues but not acyl-enzyme analogues. Conversely, DPP-4 Asp663 stabilizes dipeptidyl peptidase substrate binding and permits tight interaction with both transition state analogues. Structural modeling suggests that FAP Ala657 and DPP-4 Asp663 confer their contrasting effects on TSS by modulating the conformation of conserved residues FAP Glu204 and DPP-4 Glu206. FAP therefore requires the combined function of Ala657 and the conserved residues for endopeptidase activity.
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PMID:Ala657 and conserved active site residues promote fibroblast activation protein endopeptidase activity via distinct mechanisms of transition state stabilization. 1738 Oct 73

Management of acute and chronic pain has always been a key area of clinical research. Enkephalinase inhibitors (EIs) seem to be promising as therapeutic agents having antinociceptive action. They additionally possess anticraving, antidiarrhoeal and antidepressant actions. The antinociceptive action of EIs has been reported for over a decade however, their therapeutic potential is yet to be effectively explored. EIs may be broadly classified as endogenous and those that are obtained synthetically. Endogenous EIs include peptides like spinorphin and opiorphin. And compounds like RB 101, RB 120, RB 3007 constitute the synthetically obtained EIs. Endogenous and synthetic inhibitors enkephalin degrading enzymes have been studied in vivo using standard animal models. The potential EI targets appear to be APN (Aminopeptidase N), NEP (Neutral endopeptidase), DPP-III (Dipeptidyl peptidase). EIs possess the advantage that they lack the opioid side effects. This article reviews the mechanisms by which EIs act and elucidates the pathways involved.
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PMID:Enkephalinase inhibitors: potential agents for the management of pain. 1885 23

Porphyromonas gingivalis secretes endopeptidase gingipains, which are important virulence factors of this bacterium. Gingipains are transported across the inner membrane via the Sec system, followed by transport across the outer membrane via an unidentified pathway. The latter transport step is suggested to be mediated via a novel protein secretion pathway. In the present study, we report a novel candidate as an essential factor for the latter transport step. The PG0027 gene of P. gingivalis W83 encodes novel protein PG27. In a PG0027 deletion mutant (83K10), the activities of Arg-gingipain and Lys-gingipain were severely reduced, while the activities of secreted exopeptidases DPPIV, DPP-7, and PTP-A were unaffected. Protein localization was investigated by cell-surface biotinylation, subcellular fractionation, and immunoblot analysis. In the wild-type W83, Arg-gingipains in membrane fraction were detected as cell surface proteins. In contrast, in 83K10, Arg-gingipains were trapped in the periplasm and hardly secreted into an extracellular milieu. PG27 was suggested to be exposed to the cell surface by a cell surface biotinylation experiment; however, PG27 was detected in both inner and outer membrane fractions by subcellular fractionation experiments. Taken together, we suggest that PG27 is a unique membrane protein essential for a novel secretion pathway.
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PMID:PG27 is a novel membrane protein essential for a Porphyromonas gingivalis protease secretion system. 1918 1

Glucagon-like peptide 1 (7-36) amide (GLP-1) has been attracting considerable attention as a therapeutic agent for the treatment of type 2 diabetes. In this study, we applied a glycoengineering strategy to GLP-1 to improve its proteolytic stability and in vivo blood glucose-lowering activity. Glycosylated analogues with N-acetylglucosamine (GlcNAc), N-acetyllactosamine (LacNAc), and alpha2,6-sialyl N-acetyllactosamine (sialyl LacNAc) were prepared by chemoenzymatic approaches. We assessed the receptor binding affinity and cAMP production activity in vitro, the proteolytic resistance against dipeptidyl peptidase-IV (DPP-IV) and neutral endopeptidase (NEP) 24.11, and the blood glucose-lowering activity in diabetic db/db mice. Addition of sialyl LacNAc to GLP-1 greatly improved stability against DPP-IV and NEP 24.11 as compared to the native type. Also, the sialyl LacNAc moiety extended the blood glucose-lowering activity in vivo. Kinetic analysis of the degradation reactions suggested that the sialic acid component played an important role in decreasing the affinity of peptide to DPP-IV. In addition, the stability of GLP-1 against both DPP-IV and NEP24.11 incrementally improved with an increase in the content of sialyl LacNAc in the peptide. The di- and triglycosylated analogues with sialyl LacNAc showed greatly prolonged blood glucose-lowering activity of up to 5 h after administration (100 nmol/kg), although native GLP-1 showed only a brief duration. This study is the first attempt to thoroughly examine the effect of glycosylation on proteolytic resistance by using synthetic glycopeptides having homogeneous glycoforms. This information should be useful for the design of glycosylated analogues of other bioactive peptides as desirable pharmaceuticals.
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PMID:Chemoenzymatic synthesis of glycosylated glucagon-like peptide 1: effect of glycosylation on proteolytic resistance and in vivo blood glucose-lowering activity. 1936 Nov 94

Glucagon-like peptide-1 (GLP-1) has the ability to lower the blood glucose level, and its regulatory functions make it an attractive therapeutic agent for the treatment of type 2 diabetes. However, its rapid degradation by enzymes like dipeptidyl peptidase-IV (DPP-IV) and neutral endopeptidase (NEP) 24.11 severely compromises its effective clinical use. Whereas specific DPP-IV inhibitors have been developed, NEP 24.11 targets multiple sites in the GLP-1 sequence, which makes it difficult to block. To address this drawback, we have designed and synthesized conformationally constrained GLP-1 analogues by introducing multiple lactam bridges that stabilized both alpha-helices in the N- and C-terminal regions simultaneously. In addition to improving the receptor activation capability (up to 5-fold) by fixing the alpha-helical conformations required for optimal receptor interaction, the introduced lactam bridges provided outstanding shielding over NEP 24.11 (half-life of >96 h). These highly constrained peptides are the first examples of NEP 24.11-resistant GLP-1 analogues.
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PMID:Development of potent glucagon-like peptide-1 agonists with high enzyme stability via introduction of multiple lactam bridges. 2068 10


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