Gene/Protein Disease Symptom Drug Enzyme Compound
Pivot Concepts: Gene/Protein Disease Symptom Drug Enzyme Compound   Target Concepts: Gene/Protein Disease Symptom Drug Enzyme Compound

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Query: EC:2.7.10.1 (ERK)
95,504 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

Ig-Hepta is a member of a new subfamily of the heptahelical receptors and has an unusually long N terminus extending toward the extracellular side of the plasma membrane. Pulse-chase experiments in 293T cells using antisera specifically recognizing its N- and C-terminal regions demonstrated that Ig-Hepta is core-glycosylated cotranslationally and proteolytically processed into a two-chain form in the endoplasmic reticulum, followed by maturation of oligosaccharide chains and dimerization. The cleavage occurs at two highly conserved sites: one in a "SEA" module (a module first identified in sperm protein, enterokinase, and agrin) near the N terminus and the other in the stalk region preceding the first transmembrane span, generating approximately 20-, 130-, and 32-kDa fragments. The latter two remain tightly associated non-covalently even after cleavage as revealed by immunoprecipitation of native and myc-tagged Ig-Hepta constructs that were transiently expressed in 293T cells. The dimer consisting of four chains, (130 kDa + 32 kDa)(2), is linked by disulfide bonds. A fusion protein of the extracellular domain of Ig-Hepta and the Fc domain of immunoglobulin was found to be a good substrate of the processing enzymes and used for determining the exact cleavage sites in the SEA module and juxtamembrane stalk region.
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PMID:Cleavage of Ig-Hepta at a "SEA" module and at a conserved G protein-coupled receptor proteolytic site. 1197 29

In a previous study we showed, by transient expression studies in COS-1 cells, that the C-terminal domain of rat intestinal membrane mucin Muc3 was cleaved between glycine and serine within a GSIVV (one-letter) amino acid sequence during its residence in the endoplasmic reticulum. The extracellular domain fragment remained linked to the membrane-associated fragment by non-covalent interactions. The present study demonstrates that cleavage depends not only on the presence of the G/SIVV site (where G/S is the glycine downward arrow serine cleavage site), but also on more distant C-terminal sequences in the SEA (sea-urchin sperm protein, enterokinase and agrin) module. Inhibition of N-glycosylation by tunicamycin treatment of transfected cells did not prevent re-association of fragments, although cleavage was partially impaired, as some of the non-glycosylated, non-cleaved products were seen to accumulate in cells. Membrane targeting of the Muc3 domain and its cleavage products occurred in transfected cells and was not impaired in mutants in which the cleavage site was mutated. Targeting was also not impaired for products devoid of N-linked oligosaccharides. Our studies thus indicate that (a) cleavage within the SEA module of rat Muc3 requires participation of peptide sequences located C-terminal of and distant from the cleavage site, (b) re-association of the fragments requires the SEA module, but is independent of N-linked oligosaccharides, and (c) membrane targeting of the mucin is independent of the SEA-module-cleavage reaction.
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PMID:SEA (sea-urchin sperm protein, enterokinase and agrin)-module cleavage, association of fragments and membrane targeting of rat intestinal mucin Muc3. 1260 99

Enteropeptidase (synonym:enterokinase, EC 3.4.21.9) is a heterodimeric serine protease of the intestinal brush border that activates trypsinogen by highly specific cleavage of the trypsinogen activation peptide following the sequence (Asp)(4)-Lys. The DNA sequence encoding the light chain (catalytic subunit) of human enteropeptidase (GenBank Accession No. U09860) was synthesized from 26 oligonucleotides by polymerase chain reaction and cloned into plasmid pET-32a downstream to the gene of fusion partner thioredoxin immediately after the DNA sequence encoding enteropeptidase recognition site. The fusion protein thioredoxin/human enteropeptidase light chain was expressed in Escherichia coli BL21(DE3) strain in both soluble and insoluble forms. The soluble recombinant fusion protein failed to undergo autocatalytic cleavage and activation; however, autocatalytic cleavage and activation of recombinant human enteropeptidase light chain (L-HEP) were achieved by solubilization and renaturation of the fusion protein from inclusion bodies and the active L-HEP was purified on agarose-linked soybean trypsin inhibitor. The purified L-HEP cleaved the synthetic peptide substrate Gly-Asp-Asp-Asp-Asp-Lys-beta-naphthylamide with kinetic parameters K(m)=0.16 mM and k(cat)=115 s(-1) and small ester Z-Lys-SBzl with K(m)=140 microM, k(cat)=133 s(-1). L-HEP associated with soybean trypsin inhibitor slowly and small ester Z-Lys-SBzl cleavage was inhibited with K(i)(*)=2.3 nM. L-HEP digested thioredoxin/human epidermal growth factor fusion protein five times faster than equal activity units of bovine recombinant light chain (EKMax, Invitrogen) at the same conditions.
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PMID:Expression, purification, and characterization of human enteropeptidase catalytic subunit in Escherichia coli. 1296 50

Rat intestinal mucin Muc3 (rMuc3), like its human homologue (MUC3) and several other membrane mucins, contains a C-terminally located SEA (sea urchin sperm protein, enterokinase and agrin) module, with an intrinsic proteolytic site sequence G downward arrow SIVV (where G downward arrow S is the glycine serine cleavage site). As shown previously [Wang, Khatri and Forstner (2002) Biochem. J. 366, 623-631], expression of the C-terminal domain of rMuc3 in COS-1 cells yields a V5 epitope-tagged N-terminal glycopeptide of 30 kDa and a Myc- and His epitope-tagged C-terminal glycopeptide of 49 kDa. The present study shows that the 49 kDa membrane-anchored fragment undergoes a further cleavage reaction which decreases its size to 30 kDa. Western blotting, pulse-chase metabolic incubations, immunoprecipitation and deglycosylation with N-glycosidase F were used to detect and identify the proteolytic products. Both the first and second cleavages are presumed to facilitate solubilization of Muc3 at the apical surface of enterocytes and/or enhance the potential for Muc3 to participate in ligand-receptor and signal transduction events for enterocyte function in vivo.
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PMID:Evidence for a second peptide cleavage in the C-terminal domain of rodent intestinal mucin Muc3. 1457 8

To further elucidate the role of proteases capable of cleaving N-terminal proopiomelanocortin (N-POMC)-derived peptides, we have cloned two cDNAs encoding isoforms of the airway trypsin-like protease (AT) from mouse (MAT) and rat (RAT), respectively. The open reading frames comprise 417 amino acids (aa) and 279 aa. The mouse AT gene was located at chromosome 5E1 and contains 10 exons. The longer isoform, which we designated MAT1 and RAT1, has a simple type II transmembrane protein structure, consisting of a short cytoplasmic domain, a transmembrane domain, a SEA (63-kDa sea urchin sperm protein, enteropeptidase, agrin) module, and a serine protease domain. The human homolog of MAT1 and RAT1 is the human AT (HAT). The shorter isoform, designated MAT2 and RAT2, which contains an alternative N terminus, was formerly described in the rat as adrenal secretory serine protease (AsP) and has been shown to be involved in the processing of N-POMC-derived peptides. In contrast to the long isoform, neither MAT2 and RAT2 (AsP) contain a transmembrane domain nor a SEA domain but an N-terminal signal peptide to direct the enzyme to the secretory pathway. The C terminus, covering the catalytic triad, is identical in both isoforms. Immunohistochemically, MAT/RAT was predominantly expressed in tissues of the upper gastrointestinal and the respiratory tract-but also in the adrenal gland. Moreover, isoform-specific RT-PCR and quantitative PCR analysis revealed a complex expression pattern of the two isoforms with differences between mice and rats. These findings indicate a multifunctional role of these proteases beyond adrenal proliferation.
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PMID:The adrenal secretory serine protease AsP is a short secretory isoform of the transmembrane airway trypsin-like protease. 1469 Oct 9

We report the identification and functional analysis of a type II transmembrane serine protease encoded by the mouse differentially expressed in squamous cell carcinoma (DESC) 1 gene, and the definition of a cluster of seven homologous DESC1-like genes within a 0.5-Mb region of mouse chromosome 5E1. This locus is syntenic to a region of human chromosome 4q13.3 containing the human orthologues of four of the mouse DESC1-like genes. Bioinformatic analysis indicated that all seven DESC1-like genes encode functional proteases. Direct cDNA cloning showed that mouse DESC1 encodes a multidomain serine protease with an N-terminal signal anchor, a SEA (sea urchin sperm protein, enterokinase, and agrin) domain, and a C-terminal serine protease domain. The mouse DESC1 mRNA was present in epidermal, oral, and male reproductive tissues and directed the translation of a membrane-associated 60-kDa N-glycosylated protein with type II topology. Mouse DESC1 was synthesized in insect cells as a zymogen that could be activated by exposure to trypsin. The purified activated DESC1 hydrolyzed synthetic peptide substrates, showing a preference for Arg in the P1 position. DESC1 proteolytic activity was abolished by generic inhibitors of serine proteases but not by other classes of protease inhibitors. Most interestingly, DESC1 formed stable inhibitory complexes with both plasminogen activator inhibitor-1 and protein C inhibitor that are expressed in the same tissues with DESC1, suggesting that type II transmembrane serine proteases may be novel targets for serpin inhibition. Together, these data show that mouse DESC1 encodes a functional cell surface serine protease that may have important functions in the epidermis, oral, and reproductive epithelium.
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PMID:Mouse DESC1 is located within a cluster of seven DESC1-like genes and encodes a type II transmembrane serine protease that forms serpin inhibitory complexes. 1532 53

The synthetic gene encoding human enteropeptidase light chain (L-HEP) was cloned into plasmid pET-32a downstream from the gene of fusion partner thioredoxin immediately after the DNA sequence encoding the enteropeptidase recognition site. The fusion protein thioredoxin (Trx)/L-HEP was expressed in Escherichia coli BL21(DE3). Autocatalytic cleavage of the fusion protein and activation of recombinant L-HEP were achieved by solubilization of inclusion bodies and refolding of Trx/L-HEP fusion protein. The kinetic parameters of human and bovine enteropeptidases in the presence of different concentrations of Ca2+ and Na+ for cleavage of the specific substrate GD4K-na and nonspecific substrates such as small ester Z-Lys-SBzl and chromogenic substrates Z-Ala-X-Arg-pNA have been comparatively analyzed. It is demonstrated that positively charged ions increased the Michaelis constant (Km) for cleavage of specific substrate GD4K-na, while the catalytic constant (k(cat)) remained practically unchanged. L-HEP demonstrated secondary specificity to the chromogenic substrate Z-Ala-Phe-Arg-pNA with k(cat)/Km 260 mM(-1) x sec(-1). Enzymatic activity of L-HEP was suppressed by inhibitors of trypsin-like and cysteine (E-64), but not metallo-, amino-, or chymotrypsin-like proteinases. L-HEP was active over a broad range of pH (6-9) with optimum activity at pH 7.5, and it demonstrated high stability to different denaturing agents.
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PMID:Biochemical characterization of human enteropeptidase light chain. 1648 14

Polyserase-1 (polyserine protease-1)/TMPRSS9 (transmembrane serine protease 9) is a type II transmembrane serine protease (TTSP) that possesses unique three tandem serine protease domains. However, the physiological function of each protease domain remains poorly understood. We discovered a new splice variant of polyserase-1, termed Serase-1B, which contains 34 extra amino acids consisting a SEA module (a domain found in sea urchin sperm protein, enterokinase and agrin) adjacent to the transmembrane domain and the first protease domain with a mucin-like box at the C-terminus. The tissue distribution of this enzyme by RT (reverse transcription)-PCR analysis revealed high expression in the liver, small intestine, pancreas, testis and peripheral blood CD14+ and CD8+ cells. To investigate the role of Serase-1B, a full-length form recombinant protein was produced. Interestingly, recombinant Serase-1B was partly secreted as a soluble inactive precursor and it was also activated by trypsin. This activated enzyme selectively cleaved synthetic peptides for trypsin and activated protein C, and it was inhibited by several natural serine protease inhibitors, such as aprotinin, alpha2-antiplasmin and plasminogen activator inhibitor 1. In addition, Serase-1B efficiently converted pro-uPA (urokinase-type plasminogen activator) into active uPA and this activation was strongly inhibited by these natural inhibitors. Furthermore, this activation was also negatively regulated by glycosaminoglycans. Our results indicate that Serase-1B is a novel member of TTSPs that might be involved in uPA/plasmin-mediated proteolysis and possibly implicated in biological events such as fibrinolysis and tumour progression.
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PMID:Serase-1B, a new splice variant of polyserase-1/TMPRSS9, activates urokinase-type plasminogen activator and the proteolytic activation is negatively regulated by glycosaminoglycans. 1687 79

DESC1 was identified using gene-expression analysis between squamous cell carcinoma of the head and neck and normal tissue. It belongs to the type II transmembrane multidomain serine proteinases (TTSPs), an expanding family of serine proteinases, whose members are differentially expressed in several tissues. The biological role of these proteins is currently under investigation, although in some cases their participation in specific functions has been reported. This is the case for enteropeptidase, hepsin, matriptase and corin. Some members, including DESC1, are associated with cell differentiation and have been described as tumor markers. TTSPs belong to the type II transmembrane proteins that display, in addition to a C-terminal trypsin-like serine proteinase domain, a differing set of stem domains, a transmembrane segment and a short N-terminal cytoplasmic region. Based on sequence analysis, the TTSP family is subdivided into four subfamilies: hepsin/transmembrane proteinase, serine (TMPRSS); matriptase; corin; and the human airway trypsin (HAT)/HAT-like/DESC subfamily. Members of the hepsin and matriptase subfamilies are known structurally and here we present the crystal structure of DESC1 as a first member of the HAT/HAT-like/DESC subfamily in complex with benzamidine. The proteinase domain of DESC1 exhibits a trypsin-like serine proteinase fold with a thrombin-like S1 pocket, a urokinase-type plasminogen activator-type S2 pocket, to accept small residues, and an open hydrophobic S3/S4 cavity to accept large hydrophobic residues. The deduced substrate specificity for DESC1 differs markedly from that of other structurally known TTSPs. Based on surface analysis, we propose a rigid domain association for the N-terminal SEA domain with the back site of the proteinase domain.
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PMID:Crystal structure of the catalytic domain of DESC1, a new member of the type II transmembrane serine proteinase family. 1738 11

Post-translational modifications of the extracellular matrix receptor dystroglycan (DG) determine its functional state, and defects in these modifications are linked to muscular dystrophies and cancers. A prominent feature of DG biosynthesis is a precursor cleavage that segregates the ligand-binding and transmembrane domains into the noncovalently attached alpha- and beta-subunits. We investigate here the structural determinants and functional significance of this cleavage. We show that cleavage of DG elicits a conspicuous change in its ligand-binding activity. Mutations that obstruct this cleavage result in increased capacity to bind laminin, in part, due to enhanced glycosylation of alpha-DG. Reconstitution of DG cleavage in a cell-free expression system demonstrates that cleavage takes place in the endoplasmic reticulum, providing a suitable regulatory point for later processing events. Sequence and mutational analyses reveal that the cleavage occurs within a full SEA (sea urchin, enterokinase, agrin) module with traits matching those ascribed to autoproteolysis. Thus, cleavage of DG constitutes a control point for the modulation of its ligand-binding properties, with therapeutic implications for muscular dystrophies. We provide a structural model for the cleavage domain that is validated by experimental analysis and discuss this cleavage in the context of mucin protein and SEA domain evolution.
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PMID:SEA domain proteolysis determines the functional composition of dystroglycan. 1790 26


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