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

Enzymological studies have implicated two Ca(2+)-dependent endopeptidases in the conversion of proinsulin to insulin; a type 1 activity which cleaves on the C-terminal side of Arg31-Arg32 and a type 2 activity which cleaves C-terminally to Lys64-Arg65 in the proinsulin sequence. The possibility that these enzymes are related to the recently discovered family of mammalian subtilisin-like gene products (furin, PC2, and PC3) and the yeast propheromone-converting enzyme (KEX-2), was investigated. Degenerate oligonucleotide primers flanking the putative catalytic domain within this gene family were used in a polymerase chain reaction to amplify related sequences from rat insulinoma cDNA. One major product of 700 base pairs was obtained which was greater than 99% identical to the corresponding rat PC2 sequence. This cDNA was subcloned into the bacterial expression vector pGEX-3X to generate a recombinant protein for antibody production. Western blot analysis showed the immunoreactivity was prominent in neuroendocrine tissues as a 65-kDa protein. It was concentrated in secretory granule-enriched fractions of insulinoma tissue, where it was present as a readily solubilized monomeric protein. Deglycosylation studies using endoglycosidase H and N-glycanase showed that the 65-kDa protein was comprised of approximately 9% carbohydrate, consistent with the presence of three consensus sequences for N-linked glycosylation in rat PC2. The immunoreactivity co-eluted with the type 2 proinsulin endopeptidase on gel filtration and ion-exchange chromatography and the antisera specifically immunoprecipitated type 2 activity from insulin granule extracts. N-terminal sequence analysis of the immunoreactive protein gave two sequences which corresponded to residues 109-112 and 112-119 of rat PC2. This indicated that posttranslational processing of PC2 itself occurs C-terminally to basic amino acids to produce the mature enzyme. It is concluded that PC2 is the type 2 endopeptidase involved in proinsulin conversion. Localization of PC2 immunoreactivity to other tissues of the diffuse neuroendocrine system suggests that the type 2 endopeptidase also functions in the processing of precursor forms of other prohormones and polypeptide neurotransmitters.
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PMID:Identification of the type 2 proinsulin processing endopeptidase as PC2, a member of the eukaryote subtilisin family. 163 53

The mammalian endopeptidase, furin, is predominantly localized to the trans-Golgi network (TGN) at steady state. The localization of furin to this compartment seems to be the result of a dynamic process in which the protein undergoes cycling between the TGN and the plasma membrane. Both TGN localization and internalization from the plasma membrane are mediated by targeting information contained within the cytoplasmic domain of furin. Here, we report the results of a mutagenesis analysis aimed at identifying the source(s) of targeting information within the furin cytoplasmic domain. Our studies show that there are at least two cytoplasmic determinants that contribute to the steady-state localization and trafficking of furin. The first determinant corresponds to a canonical tyrosine-based motif, YKGL (residues 758-761), that functions mainly as an internalization signal. The second determinant consists of a strongly hydrophilic sequence (residues 766-783) that contains a large cluster of acidic residues (E and D) and is devoid of any tyrosine-based or di-leucine-based motifs. This second determinant is capable of conferring localization to the TGN as well as mediating internalization from the plasma membrane. Thus, these observations establish the existence of a novel, autonomous determinant distinct from sorting signals described previously.
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PMID:An acidic sequence within the cytoplasmic domain of furin functions as a determinant of trans-Golgi network localization and internalization from the cell surface. 758 25

Endopeptidase-24.18 (EC 3.4.24.18; meprin) is a multisubunit metallopeptidase of the astacin family. It is found in brush-border membranes of rodent kidney and human intestine. The membrane-bound enzyme is composed of alpha/beta dimers. Molecular cloning has shown that both subunits have a similar structural domain organization. Soluble alpha 2 dimers have also been observed in vivo and in transfected cells. The structures of all known alpha-subunits contain, upstream from the transmembrane domain, the sequence RXKR, which corresponds to the RXK/RR consensus sequence for specific cleavage by furin. In order to investigate the involvement of this putative cleavage site in the secretion process of endopeptidase-24,.18 alpha-subunit, we expressed in COS-1 cells rat alpha-subunits in which residues R655 or S656 (within the sequence R652PKRS656) were mutated to valine or leucine respectively. In contrast to the wild-type protein, the alpha R655V and alphaS656L mutants were not secreted in the culture medium. Moreover, when cells expressing the alpha-subunit were infected with a furin-encoding vaccinia virus, immunoblotting showed a shift of the major cell-associated form of endopeptidase-24.18 alpha-subunit from 98 kDa to 85 kDa and an increase in the amounts of secreted alpha-subunit. This shift in molecular mass was not observed with the mutant alpha-subunits. As observed for the 98 kDa species, the 85 kDa cell-associated protein was sensitive to endoglycosidase H treatment, suggesting that the proteolytic cleavage occurred in the endoplasmic reticulum or in an early Golgi compartment. Similar experiments using PACE4 and PC5 instead of furin showed that these enzymes were not able to generate the 85 kDa species. We conclude that furin is most probably the cellular enzyme involved in the proteolysis resulting in secretion of rat endopeptidase-24.18 alpha-subunit.
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PMID:Proteolytic processing of the alpha-subunit of rat endopeptidase-24.18 by furin. 762 36

To investigate the mechanisms of membrane protein localization to the Golgi complex, we have examined the intracellular trafficking of epitope-tagged forms of the mammalian endopeptidase, furin, in stably transformed rat basophilic leukemia cells. Our studies show that furin is predominantly localized to the trans-Golgi network (TGN) at steady state, with smaller amounts present in intracellular vesicles. Biochemical and morphological analyses reveal that furin is progressively delivered to a lysosomal compartment, where it is degraded. Analyses of furin deletion mutants and chimeric proteins show that the cytoplasmic domain is both necessary and sufficient for localization to the TGN in various cell types. Interestingly, deletion of most of the cytoplasmic domain of furin results in a molecule that is predominantly localized to intracellular vesicles, some of which display characteristics of lysosomes. To a lesser extent, the cytoplasmically deleted molecule is also localized to the plasma membrane. These observations suggest the existence of an additional determinant for targeting to the endosomal/lysosomal system within the lumenal and/or transmembrane domains of furin. Thus, the overall pattern of trafficking and steady state localization of furin are determined by targeting information contained within more than one region of the molecule.
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PMID:The cytoplasmic domain mediates localization of furin to the trans-Golgi network en route to the endosomal/lysosomal system. 791 93

The gamma-carboxylase recognition site in the propeptide of profactor IX signals the gamma-carboxylation of specific glutamic acid residues in the adjacent Gla domain during factor IX biosynthesis. To study posttranslational processing of the vitamin K-dependent blood coagulation factors and the properties of processing intermediates, we have isolated an incompletely processed factor IX species, profactor IX, from the medium of heterologous mammalian cells expressing the human factor IX cDNA. Profactor IX was purified by sequential immunoaffinity chromatography using antibodies specific for the propeptide and antibodies specific for the well-carboxylated factor IX species. This purified profactor IX preparation was fully gamma-carboxylated and contained the N-terminal propeptide, but it exhibited no factor IX procoagulant activity. Profactor IX was not cleaved following incubation with factor XIa. In contrast to mature factor IX, profactor IX did not demonstrate Ca(II)-dependent binding to acidic phospholipid vesicles, nor can the membrane binding surface be expressed, as detected by antibodies specific for this epitope. The propeptide of profactor IX can be removed in vitro by a specific endopeptidase, furin/PACE, yielding factor IX, which can be converted to fully active factor IXa by factor XIa and which binds normally to acidic phospholipid vesicles. These results indicate that fully gamma-carboxylated profactor IX is biologically inactive due to the presence of the propeptide.
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PMID:Profactor IX: the propeptide inhibits binding to membrane surfaces and activation by factor XIa. 794 25

The insulin proreceptor is cleaved by limited proteolysis post-translationally at an Arg-Lys-Arg-Arg site to generate its mature alpha- and beta-subunit form. An 35S-labelled insulin proreceptor substrate preparation and a 15-mer peptide substrate that mimics the amino acid sequence around and including the insulin proreceptor processing site (IRP-peptide) has revealed an endopeptidase activity that catalyses insulin proreceptor cleavage in a rat liver subcellular fraction. Under optimal conditions, normal 35S-labelled insulin proreceptor substrate processing by this fraction was quantitative. This fraction was not able to process an 35S-labelled insulin proreceptor variant substrate (where the Arg-1 of the tetrabasic cleavage site had been replaced by Ala-1), similarly to previous in vivo observations, suggesting that this endopeptidase activity has physiological relevance. Biochemical characterization of the insulin proreceptor/IRP-peptide processing revealed this rat liver endopeptidase activity to have a broad pH range (> 70% maximal activity between pH 5.5 and 10.0) and a pH optimum of pH 8-10. It was Ca(2+)-dependent activity, maximally active between 0.5 and 5 mM Ca2+ and half-maximally activated between 50 and 90 microM Ca2+. Endoproteolytic activity was not inhibited by group-specific inhibitors of serine-, cysteinyl or aspartyl proteinases or by 1,10-phenanthroline; however, EDTA and 1,2-cyclohexanediaminetetraacetic acid did inhibit the activity, but this was accounted for by Ca2+ chelation. The IRP-peptide substrate assay enabled measurement of an apparent Km of 22 microM and a Vmax of 18.6 pmol/min for this endopeptidase activity. These biochemical characteristics suggest that insulin proreceptor processing endopeptidase activity to be a legitimate member of the Kex2-related proprotein convertase family. Immunoblotting detected furin and PACE4 proteins (both members of this family) to be present in the rat liver subcellular fraction containing insulin proreceptor processing activity. Since the biochemical characteristics of the insulin proreceptor processing endopeptidase activity mostly resembled those of furin activity, it is likely that insulin proreceptor proteolytic maturation can be catalysed by furin in the liver.
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PMID:A Kex2-related endopeptidase activity present in rat liver specifically processes the insulin proreceptor. 803 79

Prohormone convertase-1 (PC1), an endopeptidase that is structurally related to the yeast subtilisin-like Kex2 gene product, has been proposed to be involved in mammalian tissue-specific prohormone processing at pairs of basic residues. To better study this enzyme, a rat somatomammotroph cell line, GH4C1, was infected with vaccinia virus recombinants of murine PC1 (mPC1) and human PC1 (hPC1). An enzymically active form of each protein was secreted into the cell medium and partially purified by anion-exchange chromatography. The 80-85 kDa enzyme was shown to be Ca(2+)-dependent and exhibited a pH optimum of 6.0 when assayed against a synthetic fluorogenic substrate, acetyl-Arg-Ser-Lys-Arg-4-methylcoumaryl-1-amide. mPC1 and hPC1 displayed identical cleavage selectivity towards a number of fluorogenic substrates, and those incorporating an Arg at the P4 site were most favoured. Synthetic peptides, encompassing the junction between the putative pro-region and the active enzyme, and between the pro-region and the biologically active parathyroid hormone, were shown to be recognized and cleaved specifically at the pair of basic residues by both enzymes. Group-specific proteinase inhibitors such as metal ion chelators and p-hydroxymercuribenzoate, but not phenylmethanesulphonyl fluoride and pepstatin, strongly inhibit the PC1-associated activity. In addition, it is shown that an enzyme activity displaying identical properties is present in the cell medium of uninfected corticotroph AtT-20 cells and that its level is increased following stimulation of secretion by the secretagogue 8-bromo cyclic AMP.
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PMID:Enzymic characterization of murine and human prohormone convertase-1 (mPC1 and hPC1) expressed in mammalian GH4C1 cells. 831 17

Prodynorphin is post-translationally processed into dynorphin B-13 and other peptides by the action of endopeptidases that cleave at pairs of basic amino acids and at single basic residues, followed by a carboxypeptidase that removes the C-terminal basic residues. To evaluate the specificity of neuropeptide processing enzymes, rat prodynorphin was transfected into BRL-3A cells, a rat liver-derived cell line which produces insulin-like growth factor II, but does not normally express prodynorphin. The transfected prodynorphin was post-translationally processed at both monobasic and dibasic cleavage sites, with the formation of dynorphin B-13 and other peptides. This finding indicates that BRL-3A cells express prodynorphin-processing enzymes. These cells were found to secrete two enzyme activities previously implicated in the processing of dynorphin, a monobasic cleaving 'dynorphin converting enzyme' and 'carboxypeptidase E', based on inhibitor sensitivities and pH optima. The dynorphin converting enzyme secreted from BRL-3A cells elutes from an anion exchange column under the same conditions as the enzyme secreted from pituitary-derived cell lines (AtT-20, GH4C1). Northern blot analysis indicates that BRL-3A cells express carboxypeptidase E mRNA in addition to mRNA encoding furin, a prohormone-processing endopeptidase. The mRNAs for two other related endopeptidases, prohormone convertase 1 and 2, were not detected on Northern blots, suggesting that these enzymes are not required for the processing of prodynorphin. The expression of carboxypeptidase E, furin, and dynorphin converting enzyme in BRL-3A cells suggests that these peptide processing enzymes are not specific for neuropeptides, but are also present in cells which process peptide growth factors.
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PMID:Processing of prodynorphin in BRL-3A cells, a rat liver-derived cell line: implications for the specificity of neuropeptide-processing enzymes. 837 74

The human cholinergic neuroepithelioma cell line SK-N-MCIXC, which expresses high levels of cholecystokinin (CCK) mRNA and secretes intact CCK into the media, was used to examine CCK processing and metabolism. Our data provide evidence for the existence of specific candidate processing enzymes in SK-N-MCIXC cells which may be involved in processing proCCK in the brain and indicate that SK-N-MCIXC cells provide a model system for studying the regulation of these enzymes. mRNAs for the intracellular processing enzymes, prohormone convertase 1 (PC1), PC2 and furin were present in SK-N-MCIXC cells. PC1 and/or PC2 and/or furin may cleave at the dibasic amino acid pairs Arg-Arg at the C-terminal part of proCCK, and Arg-X-X-Arg at the N-terminal of the CCK-58 sequence in proCCK. The SK-N-MCIXC cell line demonstrated spontaneous and regulated release of CCK and large amounts of CCK-precursors, as measured with region specific radioimmunoassays coupled to high performance liquid chromatography. Storage granules containing glycine-extended CCK were shown in SK-N-MCIXC cells using indirect immunofluorescence. The extracellularly localized CCK-metabolizing enzyme, neutral endopeptidase 24.11 (EC 3.4.24.11), was present in membranes from both SK-N-MCIXC cells and in intact slices of rat cerebral cortex. The rat cerebral cortex is a brain region known to be rich in CCK. The SK-N-MCIXC cell line provides an in vitro model to study the regulation of CCK synthesis and metabolism in neuronal systems since it contains the storage granules, mRNA, intact peptide, and complement of enzymes necessary for biosynthesis and metabolism of CCK.
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PMID:Processing, release and metabolism of cholecystokinin in SK-N-MCIXC cells. 841 49

We describe a new member of the receptor protein tyrosine phosphatase family, R-PTP-kappa, cDNA cloning predicts that R-PTP-kappa is synthesized from a precursor protein of 1,457 amino acids. Its intracellular domain displays the classical tandemly repeated protein tyrosine phosphatase homology, separated from the transmembrane segment by an uncharacteristically large juxta-membrane region. The extracellular domain of the R-PTP-kappa precursor protein contains an immunoglobulin-like domain and four fibronectin type III-like repeats, preceded by a signal peptide and a region of about 150 amino acids with similarity to the Xenopus A5 antigen, a putative neuronal recognition molecule (S. Takagi, T. Hsrata, K. Agata, M. Mochii, G. Eguchi, and H. Fujisawa, Neuron 7:295-307, 1991). Antibodies directed against the intra- and extracellular domains reveal that the R-PTP-kappa precursor protein undergoes proteolytic processing, following which both cleavage products remain associated. By site-directed mutagenesis, the likely cleavage site was shown to be a consensus sequence for cleavage by the processing endopeptidase furin, located in the fourth fibronectin type III-like repeat. In situ hybridization analysis indicates that expression of R-PTP-kappa in the central nervous system is developmentally regulated, with highest expression seen in actively developing areas and, in the adult, in areas capable of developmental plasticity such as the hippocampal formation and cerebral cortex. The mouse R-PTP-kappa gene maps to chromosome 10, at approximately 21 centimorgans from the centromere.
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PMID:Cloning and characterization of R-PTP-kappa, a new member of the receptor protein tyrosine phosphatase family with a proteolytically cleaved cellular adhesion molecule-like extracellular region. 847 52


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