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)

Furin is a dibasic endopeptidase responsible for the proteolytic maturation of many precursor proteins in the secretory and endocytic pathways of mammalian cells. The levels of furin expression in most cells are very low, and this has hampered attempts to identify the intracellular compartments in which endogenous furin is localized. We have used a specific antibody reagent to a sequence in the carboxy terminus of furin to perform immunofluorescent staining of mammalian cell lines. This antibody was sensitive enough to detect staining for furin in various cell lines. For the most part, furin staining was confined to a juxtanuclear structure characteristic of the Golgi complex. Analyses by video microscopy and confocal microscopy showed that the distribution of furin was distinct from that of mannosidase II, a marker of the Golgi stack, and most closely resembled that of TGN38, a marker of the trans-Golgi network. Therefore, our results suggest that endogenous furin is predominantly localized to the area of the Golgi complex, most likely within the trans-Golgi network.
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PMID:Localization of endogenous furin in cultured cell lines. 901 Apr 63

The mammalian endopeptidase furin is a type 1 integral membrane protein that is predominantly localized to the TGN and is degraded in lysosomes with a t1/2 = 2-4 h. Whereas the localization of furin to the TGN is largely mediated by sorting signals in the cytosolic tail of the protein, we show here that targeting of furin to lysosomes is a function of the luminal domain of the protein. Inhibition of lysosomal degradation results in the accumulation of high molecular weight aggregates of furin; aggregation is also dependent on the luminal domain of furin. Temperature and pharmacologic manipulations suggest that furin aggregation occurs in the TGN and thus precedes delivery to lysosomes. These findings are consistent with a model in which furin becomes progressively aggregated in the TGN, an event that leads to its transport to lysosomes. Our observations indicate that changes in the aggregation state of luminal domains can be potent determinants of biosynthetic targeting to lysosomes and suggest the possible existence of quality control mechanisms for disposal of aggregated proteins in compartments of the secretory pathway other than the endoplasmic reticulum.
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PMID:Aggregation as a determinant of protein fate in post-Golgi compartments: role of the luminal domain of furin in lysosomal targeting. 941 68

The ADAMs (a disintegrin and metalloprotease) are a family of multidomain proteins with structural homology to snake venom metalloproteases. We recently described the cloning and sequencing of human ADAM 12 (meltrin alpha). In this report we provide evidence that the metalloprotease domain of ADAM 12 is catalytically active. We used the trapping mechanism of alpha2-macroglobulin to assay for protease activity of wild-type and mutant ADAM 12 proteins produced in a COS cell transfection system. We found that ADAM 12 is synthesized as a zymogen, with the prodomain maintaining the metalloprotease in a latent form, probably by means of a cysteine switch. The zymogen could be activated chemically by alkylation with N-ethylmaleimide. Cleavage of the prodomain at a site for a furin-like endopeptidase resulted in an ADAM 12 protein with proteolytic activity. The protease activity was sensitive to inhibition by 1,10-phenanthroline and could be eliminated by mutation of the critical glutamate residue at the active site. The demonstration that the ADAM 12 metalloprotease domain is functional may have important implications for future studies that explore the role of ADAM 12 protein in development and disease.
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PMID:Human ADAM 12 (meltrin alpha) is an active metalloprotease. 964 63

Cellular disintegrin and metalloproteinases (ADAMs) are a family of genes with a sequence similar to the snake venom metalloproteinases and disintegrins. ADAMTS-1 is a unique ADAM family protein with respect to the presence of thrombospondin type I motifs and the capacity to bind to the extracellular matrix. Because ADAMTS-1 has a potential zinc-binding motif in the metalloproteinase domain, we examined in this study whether ADAMTS-1 is an active metalloproteinase by means of the proteinase trapping mechanism of alpha2-macroglobulin. We found that the soluble type of ADAMTS-1 protein is able to form a covalent-binding complex with alpha2-macroglobulin. Furthermore, the point mutation within the zinc-binding motif of ADAMTS-1 protein eliminates its capacity to bind to alpha2-macroglobulin. These data demonstrate that the metalloproteinase domain of ADAMTS-1 is catalytically active. In addition, we showed that the removal of the pro-domain from the ADAMTS-1 precursor is impaired in the furin-deficient cell line, LoVo, and that the processing ability of the cells is restored by the co-expression of the furin cDNA. These data provide evidence that the ADAMTS-1 precursor is processed in vivo by furin endopeptidase in the secretory pathway. Consequently, ADAMTS-1 is an active metalloprotease that is associated with the extracellular matrix. This study strongly suggests that ADAMTS-1 may play a role in the inflammatory process through its protease activity.
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PMID:ADAMTS-1 is an active metalloproteinase associated with the extracellular matrix. 1037

Success in controlling hyperglycemia in type I diabetics will require a restoration of basal insulin. To this end, three plasmid DNAs (pDNA) encoding preproinsulin were compared for constitutive expression and processing to insulin in nonendocrine cells in vitro. The pDNAs were designed to express rat proinsulin I (VR-3501), rat proinsulin I with the B10 aspartic acid point mutation (VR-3502), and a derivative of VR-3502 with a furin cleavage site added at the B-chain and C-peptide junction (VR-3503). Cells transfected with VR-3501 or VR-3502 were able to secrete only proinsulin, whereas transfection with VR-3503 yielded 30-70% mature insulin, which could be increased to >99% by cotransfection with a furin expression plasmid (VR-3505). The insulin produced was biologically active. The bilateral injection of 100 microg of VR-3502 plasmid into the tibialis anterior muscles of mice on two consecutive days yielded, on average, several hundred picograms of heterologous proinsulin per milliliter of serum. In BALB/c mice, serum proinsulin peaked 7-14 days postinjection and declined to preinjection levels by days 21-28. In athymic nude mice, serum proinsulin was sustained for at least 6 weeks. The therapeutic efficacy of delivering insulin via muscle injection of pDNA was evaluated in athymic nude mice made diabetic with the beta cell toxin streptozotocin (STZ). All animals given control DNA died within 1 week of receiving STZ while 40% of the mice coinjected with plasmids VR-3503 and VR-3505 lived through the duration of the 4-week experiment. Muscles of the surviving animals contained 17-100 ng of immune-reactive insulin (IRI), 86-94% of which was mature insulin. The results suggest that heterologous insulin made in muscle increased the survival rate. We propose that insulin plasmid expression in skeletal muscle may be a valid approach to basal insulin delivery. The feasibility of plasmid DNA-based delivery of basal insulin was investigated. An expression system consisting of pDNAs encoding a selectively mutated rat preproinsulin and mouse furin was developed and characterized in vitro and in vivo. When injected with preproinsulin pDNA, the mouse tibialis anterior muscle expressed and released proinsulin into serum at levels comparable to normal basal insulin in rodents. These heterologous proinsulin levels were sustained for several weeks in immune-compromised nondiabetic mice. Mouse muscle coinjected with a pDNA encoding the endopeptidase furin and a pDNA encoding a pre-proinsulin modified to contain two furin cleavage sites produced fully processed insulin. This muscle-made insulin appears to have contributed to the survival of mice treated with a highly diabetogenic dose of streptozotocin, a beta cell toxin. The results demonstrate that skeletal muscle is able to express and deliver therapeutic insulin from plasmid DNA.
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PMID:Insulin delivery with plasmid DNA. 1056 91

Because of their roles in controlling the activity of several bio-active peptides, members of the neprilysin family of zinc metallopeptidases have been identified as putative targets for the design of therapeutic agents. Presently, six members have been reported, these are: neprilysin, endothelin-converting enzyme (ECE)-1 and ECE-2, the Kell blood group protein, PHEX (product of the phosphate-regulating gene with homologies to endopeptidase on the X chromosome) and X-converting enzyme (XCE). In order to identify new members of this important family of peptidases, we designed a reverse transcriptase-PCR strategy based on conserved amino acid sequences of neprilysin, ECE-1 and PHEX. We now report the cloning from mouse testis of a novel neprilysin-like peptidase that we called NL1. NL1 is a glycoprotein that, among the members of the family, shows the strongest sequence identity with neprilysin. However, in contrast with neprilysin and other members of the family which are type II integral membrane proteins, NL1 was secreted when expressed in cultured mammalian cells, likely due to cleavage by a subtilisin-like convertase at a furin-like site located 22 amino acid residues in the C-terminus of the transmembrane domain. The recombinant enzyme exhibited neprilysin-like peptidase activity and was efficiently inhibited by phosphoramidon and thiorphan, two inhibitors of neprilysin. Northern blot analysis and in situ hybridization showed that NL1 mRNA was found predominantly in testis, specifically in round and elongated spermatids. This distribution of NL1 mRNA suggests that it could be involved in sperm formation or other processes related to fertility.
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PMID:Molecular cloning and biochemical characterization of a new mouse testis soluble-zinc-metallopeptidase of the neprilysin family. 1074 71

The majority of CTL epitopes are derived from intracellular proteins that are degraded in the cytoplasm by proteasomes into peptides that are transported into the endoplasmic reticulum by the TAP complex. These peptides can be further processed into the optimal size (8-10 residues) for binding with nascent MHC class I molecules, generating complexes that are exported to the cell surface. Proteins or peptides containing CTL epitopes can be introduced into the cytoplasm of APCs by linking them to membrane-translocating Trojan carriers allowing their incorporation into the MHC class I Ag-processing pathway. The present findings suggest that these "Trojan" Ags can be transported into the endoplasmic reticulum in a TAP-independent way where they are processed and trimmed into CTL epitopes. Furthermore, processing of Trojan Ags can also occur in the trans-Golgi compartment, with the participation of the endopeptidase furin and possibly with the additional participation of a carboxypeptidase. We believe that these findings will be of value for the design of CTL-inducing vaccines for the treatment or prevention of infectious and malignant diseases.
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PMID:TAP-independent presentation of CTL epitopes by Trojan antigens. 1139 Apr 50

Members of the neutral endopeptidase (NEP, also known as MME for membrane metallo-endopeptidase in the Human Gene Nomenclature database) family play significant roles in pain perception, arterial pressure regulation, phosphate metabolism, and homeostasis. In this paper, we report the cloning of a new human member of the NEP family that we named MMEL2 for membrane metallo-endopeptidase-like 2. The MMEL2 protein has the structural characteristics of type II transmembrane proteins, although the presence of a furin-like cleavage site in the ectodomain suggests that it may be released into the medium following proteolytic cleavage. The MMEL2 protein contains the zinc-binding consensus sequence HEXXH and all the residues known to be essential for the enzymatic activity of other members of the family. The MMEL2 mRNA was detected predominantly in testis, but weak expression also was observed in brain, kidney, and heart. The human MMEL2 gene was mapped to 1p36 by fluorescence in situ hybridization. It will be important to test whether MMEL2 defects are associated with diseases such as hereditary motor sensory neuropathy 2A, Schwartz-Jampel-Aberfeld syndrome, or neuroblastoma, which all map to this locus.
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PMID:Molecular cloning, tissue distribution, and chromosomal localization of MMEL2, a gene coding for a novel human member of the neutral endopeptidase-24.11 family. 1156 Jul 81

This work generated many truncated proteins and Glu(385) to Ala (E(385)/A) mutants of the human metalloproteinase and thrombospondin 1 (METH-1 or ADAMTS1) and specific antibodies. METH-1 was an active endopeptidase and both the metalloproteinase and the disintegrin/cysteine-rich domains were required for the proteinase activity. A point mutation at the zinc-binding site (E(385)/A) abolished the catalytic activity. METH-1 protein function may be modulated through proteolytic cleavage at multiple sites. One 135 kDa species had an NH(2)-terminal sequence of L(33)GRPSEEDEE. A species at 115 kDa and some other protein bands began with F(236)VSSHRYV(243), indicating that METH-1 proenzyme might be activated by a proprotein convertase such as furin by cleaving the R(235)-F(236) peptide bond. This cleavage was not an autocatalytic process since the E(385)/A mutants were also processed. Furthermore, a 52 kDa band with an NH(2)-terminal sequence of L(800)KEPLTIQV resulted from the digestion between the first and the second thrombospondin 1-like motifs in the spacer region of the extracellular matrix-binding domains.
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PMID:Protein engineering and properties of human metalloproteinase and thrombospondin 1. 1205 26

The imaginal discs of Drosophila melanogaster give rise to the adult epidermis during metamorphosis. During this developmental period several peptidase genes are expressed in disc cells, but there is a paucity of biochemical information regarding substrate specificity. We have used peptides and peptidyl 7-amino-4-methylcoumarin (AMC) substrates to detect several peptidases either positioned on the surface of wing discs or secreted by the imaginal cells. Using [Leu(5)]enkephalin as a substrate, a captopril sensitive dipeptidyl carboxypeptidase (angiotensin I-converting enzyme) and an amastatin-sensitive aminopeptidase were detected as prominent activities associated with intact discs. The formation of [Leu(5)]enkephalin-derived Phe was attributed to the concerted action of the D. melanogaster angiotensin I-converting enzyme (Ance) and a dipeptidase. The disc Ance also showed endopeptidic activity towards locust tachykinin-1 (LomTK-I) by cleaving the Gly-Val peptide bond, but this enzyme was not the sole endopeptidase activity associated with discs. Complete inhibition of the endopeptidic hydrolysis of the LomTK-1 by a disc homogenate required a combination of captopril and the neprilysin inhibitor, phosphoramidon, providing biochemical evidence for a neprilysin-like peptidase, in addition to Ance, in imaginal discs of D. melanogaster. Peptidyl AMC substrates for furin, prohormone convertase and tryptase provided evidence for trypsin-like serine endopeptidases in addition to the metalloendopeptidases. We conclude that imaginal discs are endowed with a variety of peptidases from different families that together are capable of hydrolyzing a broad range of peptides and proteins. Some of these peptidases might be responsible for the metabolic activation/inactivation of signaling peptides, as well as being involved in the production of dipeptides and free amino acids required for protein synthesis and osmotic balance during adult morphogenesis.
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PMID:Extracellular peptidases of imaginal discs of Drosophila melanogaster. 1243 39


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