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:3.4.21.4 (trypsin)
42,187 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

In order to shed further light on the potential role of mast cells during tissue turnover, we have investigated the number of mast cells containing only tryptase and those storing both tryptase and chymase by enzyme histochemistry in normal versus healing skin. Furthermore, we have studied the in vitro effect of these enzymes on the mitogenesis of subconfluent quiescent fibroblast and HaCaT keratinocyte cultures, using flowcytometric DNA analysis. Chymase-containing mast cell numbers were markedly decreased in scars (P<0.001), whereas the overall number of tryptase-containing mast cells was not decreased, although these cells were smaller and stained more faintly in scars. Chymase (5 to 300 mU/ml) induced a marked, dose-dependent in vitro mitogenic response in 3T3 fibroblasts, whereas the effects of tryptase, at up to 60 nM, were only moderate, compared to the known fibroblast mitogens EGF, TGF-alpha, alpha-thrombin and trypsin at optimal concentrations. Coincubation of either protease with EGF or alpha-thrombin had additive effects. In contrast to fibroblasts, keratinocytes showed only minor mitogenic responses to tryptase and chymase, also in comparison to other known mitogenic stimuli, and responses to EGF and alpha-thrombin were inhibited on costimulation of cells with the proteases. These findings document for the first time a potential role of mast cell chymase in connective tissue repair, with tryptase being less active on fibroblasts, and with inhibitory effects of both mast cell proteases on keratinocytes.
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PMID:Mast cell chymase and tryptase during tissue turnover: analysis on in vitro mitogenesis of fibroblasts and keratinocytes and alterations in cutaneous scars. 1038 36

Immobilized proteolytic enzyme cartridges were used to rapidly digest neu differentiation factor EGF domain in order to obtain improved peptide maps useful for assignment of disulfide linkages. The procedure described here involves an on-line digestion of proteins using immobilized trypsin and endoproteinase Glu-C cartridges connected in series, followed by on-line RP-HPLC separation of the peptides. The entire process can be automated using a commercially available workstation; and the total time required for both proteolytic digestion and the HPLC separation can be shortened to within 1 h. Using these immobilized columns, we demonstrated that disulfide structure assignment of the EGF domains of recombinant human neu differentiation factor can be performed by isolation of individual disulfide-containing peptides followed by assignment of disulfide linkages with prompt fragmentation of peptides using matrix-assisted laser desorption/ionization time-of-flight mass spectrometry. The use of immobilized protease cartridges in tandem eliminates undesirable digestion artifacts associated with longer digestion time and higher protease-to-substrate ratio and results in the development of a reproducible and high quality peptide map.
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PMID:Peptide map procedure using immobilized protease cartridges in tandem for disulfide linkage identification of neu differentiation factor epidermal growth factor domain. 1067 Jul 17

Mouse SPACR cDNA was cloned by screening a mouse retina cDNA library using a PCR probe derived from human SPACR cDNA. Mouse SPACR cDNA comprises 3675 bp containing an open reading frame coding for 742 amino acids. Multitissue Northern blot analysis and in situ hybridization studies indicate that SPACR expression is restricted to retinal photoreceptors. The SPACR core protein was identified with Western blotting following SDS-PAGE with a SPACR C-terminal peptide polyclonal antibody and a chondroitin-6-sulfate Deltadisaccharide monoclonal antibody. The 150 kD immunopositive band was isolated, digested with trypsin and the peptides analysed by MALDI mass spectroscopy. Peptide mass mapping confirmed the identity of the 150 kD immunopositive band to be mouse SPACR core protein. Alignment comparisons of the deduced amino acid sequence of mouse and human SPACR show 64% homology. Like SPACR in the human interphotoreceptor matrix, the mouse orthologue contains a large central mucin-like domain flanked by consensus sites for N-linked oligosaccharide attachment, one EGF-like domain and four hyaluronan-binding motifs. Unlike human SPACR, which contains no conventional consensus sites for glycosaminoglycan attachment, mouse SPACR contains three. Recent biochemical studies of human and mouse SPACR protein indicate that this novel interphotoreceptor matrix molecule is a glycoprotein in human and a proteoglycan in the mouse. The presence of consensus sites for glycosaminoglycan attachment in the deduced sequence of mouse SPACR and the absence of these sites in human SPACR provide molecular verification of our biochemical results, suggesting that differences in post-translational modifications of SPACR may be important in SPACR function in foveate and non-foveate retinas.
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PMID:SPACR in the interphotoreceptor matrix of the mouse retina: molecular, biochemical and immunohistochemical characterization. 1099 55

Human fetuin, [alpha2-Heremans Schmid Glycoprotein (alpha2-HSG)], is a natural inhibitor of insulin receptor tyrosine kinase activity (IR-TKA). Previously, we have demonstrated that alpha2-HSG inhibits the mitogenic pathway without affecting the metabolic arm of insulin signal transduction. In this study, we demonstrate the time-course and specificity of inhibition, its interaction with IR and probable physiological role. In intact rat1 fibroblasts overexpressing the human insulin receptor (HIRc B), incubation of recombinant human alpha2-HSGbac (1.8 microM) inhibited insulin-induced IR autophosphorylation by over 80%. This inhibitory effect of alpha2-HSGbac on insulin-induced IR autophosphorylation was blunted by half in 60 min. Interestingly, alpha2-HSGbac at similar concentrations (0.9 or 1.8 microM), had no effect on EGF- or IGF-I-induced cognate receptor autophosphorylation. Anti-alpha2-HSG immunoprecipitates of alpha2-HSGbac-treated HIRc B cell lysates demonstrated the presence of IR. Our data suggest that alpha2-HSGbac preferentially interacts with the activated IR. To further characterize the site(s) of interaction, the effect of alpha2-HSGbac on trypsin-treated IR autophosphorylation was studied. Trypsin-treatment of intact HIRc B cells results in proteolysis of the IR alpha-chain and constitutive activation of IR-TKA. We demonstrate that alpha2-HSGbac (0.1 microM) completely inhibited trypsin-activated IR autophosphorylation and TKA in vitro indicating that this effect was not mediated by its interaction with the proximal 576 amino acid residues of the IR alpha-subunit. The physiological relevance of these observations was explored by characterizing the effects of alpha2-HSG injection in rats. Alpha2-HSGbac (2 microM), acutely injected through the portal vein of normal rats, inhibited insulin-stimulated IR autophosphorylation and IRS-1 phosphorylation in liver and hindlimb muscle. Taken together our results suggest that alpha2-HSG, by interacting with IR, specifically inhibits insulin-stimulated IR autophosphorylation and may play a physiological role in the regulation of insulin signaling.
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PMID:Alpha2-HSG, a specific inhibitor of insulin receptor autophosphorylation, interacts with the insulin receptor. 1102 61

Factor Xa plays a critical role in the formation of blood clots. This serine protease catalyzes the conversion of prothrombin to thrombin, the first joint step that links the intrinsic and extrinsic coagulation pathways. There is considerable interest in the development of factor Xa inhibitors for the intervention in thrombic diseases. This paper presents the structure of the inhibitor ZK-807834, also known as CI-1031, bound to factor Xa and provides the details of the protein purification and crystallization. Results from mass spectrometry indicate that the factor Xa underwent autolysis during crystallization and the first EGF-like domain was cleaved from the protein. The crystal structure of the complex shows that the amidine of ZK-807834 forms a salt bridge with Asp189 in the S1 pocket and the basic imidazoline fits snugly into the S4 site. The central pyridine ring provides a fairly rigid linker between these groups. This rigidity helps minimize entropic losses during binding. In addition, the structure reveals new interactions that were not found in the previous factor Xa/inhibitor complexes. ZK-807834 forms a strong hydrogen bond between an ionized 2-hydroxy group and Ser195 of factor Xa. There is also an aromatic ring-stacking interaction between the inhibitor and Trp215 in the S4 pocket. These interactions contribute to both the potency of this compound (K(I) = 0.11 nM) and the >2500-fold selectivity against homologous serine proteases such as trypsin.
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PMID:Preparation, characterization, and the crystal structure of the inhibitor ZK-807834 (CI-1031) complexed with factor Xa. 1102 32

Characterization of the major human milk fat globular membrane proteins was carried out using proteomic techniques comprising two-dimensional polyacrylamide gel electrophoresis, followed by in situ PNGase F and trypsin digestion. Matrix-assisted laser desorption/ionization quadrupole time-of-flight and electrospray ionization mass spectrometry identified seven major protein components: alpha-lactalbumin, lysozyme precursor, beta-casein, clusterin, lactotransferrin, polymeric immunoglobulin receptor precursor, and human milk fat globule EGF-factor 8 protein. Sequence information on the protein-associated glycans was determined by matrix-assisted laser desorption-ionization quadrupole time-of-flight hybrid mass spectrometry. This glycan analysis revealed interesting fucosylation branching patterns which may be influential in maternal protection of the newborn against bacterial and viral pathogenic attack.
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PMID:Use of proteomic methodology for the characterization of human milk fat globular membrane proteins. 1181 2

Fertilization is a precisely controlled process involving many gamete molecules in sperm binding to and penetration through the extracellular matrix of the egg. After sperm bind to the extracellular matrix (vitelline coat), they undergo the acrosome reaction which exposes and partially releases a lytic agent called "lysin" to digest the vitelline coat for the sperm penetration. The vitelline coat sperm lysin is generally a protease in deuterostomes. The molecular mechanism of the actual degradation of the vitelline coat, however, remains poorly understood. In order to understand the lysin system, we have been studying the fertilization mechanism in ascidians (Urochordata) because we can obtain large quantities of gametes which are readily fertilized in the laboratory. Whereas ascidians are hermaphrodites, which release sperm and eggs simultaneously, many ascidians, including Halocynthia roretzi, are strictly self-sterile. Therefore, after sperm recognize the vitelline coat as nonself, the sperm lysin system is thought to be activated. We revealed that two sperm trypsin-like proteases, acrosin and spermosin, the latter of which is a novel sperm protease with thrombin-like substrate specificity, are essential for fertilization in H. roretzi. These molecules contain motifs involved in binding to the vitelline coat. We found that the proteasome rather than trypsin-like proteases has a direct lytic activity toward the vitelline coat. The target for the ascidian lysin was found to be a 70-kDa vitelline coat component called HrVC70, which is made up of 12 EGF-like repeats. In addition to the proteasome system, the ubiquitination system toward the HrVC70 was found to be necessary for ascidian fertilization. In this review, I describe recent progress on the structures and roles in fertilization of the two trypsin-like proteases, acrosin and spermosin, and also on the novel extracellular ubiquitin-proteasome system, which plays an essential role in the degradation of the ascidian vitelline coat.
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PMID:Ascidian sperm lysin system. 1201 76

This is a study on the cultivation condition in vitro and differentiation of neural stem cells from human embryonic brain in order to find a way to get purified multipotential neural stem cells. The single cells was derived from the three-month embryonic brain digested with trypsin, some cells was frozen, the other cells were expanded with EGF and bFGF, the single-cell-clone was obtained by the way of limited dilution, and the serum was used to induce the cells differentiation. The cells were detected with the method of immunohistochemistry. The results showed that a lot of neurospheres could be seen in the presence of mitogens (both EGF and bFGF) and serum could induce neural stem cells to differentiate into neurons, astrocytes, and oligodendrocytes. These indicate that the survival and proliferation of neural stem cells rely on the cooperation of EGF and bFGF. The neural stem cells can also be harvested from the frozen cells.
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PMID:[Isolation, cultivation and identification of neural stem cell from human embryonic CNS]. 1222 96

The exact role profilin plays in cell migration is not clear. In this study, we have evaluated the effect of overexpression of profilin on the migration of breast cancer cells. Overexpression was carried out by stably expressing GFP-profilin in BT474 cells. It was observed that even a moderate level of overexpression of profilin significantly impaired the ability of BT474 cells to spread on fibronectin-coated substrate and migrate in response to EGF. GFP-profilin expressing cells also showed increased resistance to detachment in response to trypsin and increased tyrosine phosphorylation of focal adhesion kinase (FAK) and paxillin compared to the parental and GFP-expressing (control) cell lines. These results suggest that perturbation of profilin levels may offer a good strategy for controlling the metastatic potential of breast cancer cells.
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PMID:Overexpression of profilin reduces the migration of invasive breast cancer cells. 1469 48

Several lines of evidence suggest that tumor-derived trypsin contributes to the growth and invasion of cancer cells. We have recently shown that trypsin is a potent growth factor for colon cancer cells through activation of the G protein-coupled receptor protease-activated receptor 2 (PAR2). Here, we analyzed the signaling pathways downstream of PAR2 activation that lead to colon cancer cell proliferation in HT-29 cells. Our data are consistent with the following cascade of events upon activation of PAR2 by the serine protease trypsin or the specific PAR2-activating peptide (AP2): (i) a matrix metalloproteinase-dependent release of transforming growth factor (TGF)-alpha, as demonstrated with TGF-alpha-blocking antibodies and measurement of TGF-alpha in culture medium; (ii) TGF-alpha-mediated activation of epidermal growth factor receptor (EGF-R) and subsequent EGF-R phosphorylation; and (iii) activation of ERK1/2 and subsequent cell proliferation. The links between these events are demonstrated by the fact that stimulation of cell proliferation and ERK1/2 upon activation of PAR2 is reversed by the metalloproteinase inhibitor batimastat, TGF-alpha-neutralizing antibodies, EGF-R ligand binding domain-blocking antibodies, and the EGF-R tyrosine kinase inhibitors AG1478 and PD168393. Therefore, transactivation of EGF-R appears to be a major mechanism whereby activation of PAR2 results in colon cancer cell growth. By using the Src tyrosine kinase inhibitor PP2, we further showed that Src plays a permissive role for PAR2-mediated ERK1/2 activation and cell proliferation, probably acting downstream of the EGF-R. These data explain how trypsin exerts robust trophic action on colon cancer cells and underline the critical role of EGF-R transactivation.
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PMID:Protease-activated receptor 2 in colon cancer: trypsin-induced MAPK phosphorylation and cell proliferation are mediated by epidermal growth factor receptor transactivation. 1501 Apr 75


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