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)

Fc epsilon RI cross-linkage in mast cells results in release of granule-associated mediators, such as histamine and proteases, as well as the production of numerous cytokines, including IL-6. Mast cells have been increasingly implicated in inflammatory processes where explosive degranulation is not commonly observed. Here, we show that IL-1 stimulates secretion of IL-6 without release of the granule-associated protease tryptase in normal human umbilical cord blood-derived mast cells (hCBMCs). IL-6 secretion stimulated by IL-1 in hCBMCs is potentiated by priming with IL-4 and reflects the higher levels of IL-6 secreted from human leukemic mast cell line (HMC-1). Stimulating HMC-1 cells by both IL-1 and TNF-alpha results in synergistic secretion of IL-6. IL-6 is de novo synthesized, as its secretion is blocked by inhibitors of transcription or protein synthesis. IL-1 does not increase intracellular calcium ion levels in either hCBMCs or HMC-1 cells, and IL-6 stimulation proceeds in the absence of extracellular calcium ions. Ultrastructural Immunogold localization shows that IL-6 is excluded from the secretory granules and is compartmentalized in 40- to 80-nm vesicular structures. Selective secretion of IL-6 from mast cells appears distinct from degranulation and may contribute to the development of inflammation, where the importance of IL-6 has been recognized.
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PMID:IL-1 induces vesicular secretion of IL-6 without degranulation from human mast cells. 1456 62

Inflammatory foci are rich in proteases released by neutrophils (serine proteases) and macrophages (metalloproteases). These enzymes can degrade extracellular matrix proteins and cell membrane bound proteins thus contributing to the development and progression of inflammatory reaction. In this study we have investigated the influence of collagenase (metalloprotease) and trypsin (serine protease) on murine resident and oil-induced peritoneal macrophages (Mf). Short in vitro treatment of Mf, not affecting cell viability, significantly reduced the release of reactive oxygen intermediates (ROIs) and at the same time triggered the increase of IL-6 production and to lesser extent of TNF-alpha production. Both these effects were dependent on enzyme concentration used and were particularly well pronounced in resident macrophages. In addition both enzymes cleaved a number of cell-membrane molecules, including CD23, CD14, CD95L, and Mac-3. We hypothesize that the enzymatic digestion of certain Mf surface receptor proteins in inflammatory foci may be responsible for modification of cell behaviour either by preventing the generation of specific signal or alternatively by delivering a mock substitute signal to the cell interior. In effect inhibition of ROIs production limits their destructive effects and the increase in the secretion of IL-6 stimulates the synthesis of acute phase proteins and triggers other anti-inflammatory mechanisms thus directing Mf present in inflammatory foci into regulatory pathway rather than allowing them to perform solely the effector function.
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PMID:Modulation of macrophage activity by proteolytic enzymes. Differential regulation of IL-6 and reactive oxygen intermediates (ROIs) synthesis as a possible homeostatic mechanism in the control of inflammation. 1476 Sep 41

Type 1, or cellular, immune response is characterized by overproduction of TNF-alpha, IFN-gamma, IL-1, IL-2 and IL-8 and is the underlying immune mechanism of psoriasis, alopecia areata, rheumatoid arthritis, Crohn's disease, multiple sclerosis, insulin-dependent diabetes mellitus and experimental autoimmune uveitis (EAU). Type 2 immune response is seen in antibody-mediated autoimmune diseases. Based on the pharmacokinetic effects of cetirizine and allopurinol, this paper introduces these two safe and inexpensive drugs as novel potential agents against cell-mediated autoimmune disorders. Cetirizine, supposed to inhibit DNA binding activity of NF-kappa B, inhibits the expression of adhesion molecules on immunocytes and endothelial cells and the production of IL-8 and LTB4, two potent chemoattractants, by immune cells. It induces the release of PGE2, a suppressor of antigen presentation and MHC class II expression, from monocyte/macrophages and reduces the number of tryptase positive mast cells in inflammation sites. Tryptase is a chemoattractant, generates kinins from kininogen, activates mast cells, triggers maturation of dendritic cells and stimulates the release of IL-8 from endothelial cells and the production of Th1 lymphokines by mononuclear immunocytes. Allopurinol is a free radical scavenger, suppresses the production of TNF-alpha and downregulates the expression of ICAM-1 and P2X(7) receptors on monocyte/macrophages. ICAM-1 serves as a ligand for LFA-1 (on T lymphocytes), allowing proper antigen presentation. P2X(7) receptors are thought to be involved in IL-1beta release, mitogenic stimulation of T lymphocytes and the probable cytoplasmic communication between macrophages and lymphocytes at inflammation sites. Allopurinol was markedly more effective than prednisolone in treating experimental autoimmune uveitis and in combination with cyclosporine suppressed the inflammatory reaction of this condition more effectively than either agent alone. As allopurinol is a competitive inhibitor of xanthine oxidase and decreases serum levels of uric acid, which is protective against multiple sclerosis, it should preferably be coadministered with uric acid precursors in the treatment of this condition. Cetirizine and allopurinol may prove of benefit in the treatment of various cellular autoimmune disorders.
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PMID:Cetirizine and allopurinol as novel weapons against cellular autoimmune disorders. 1503 12

Stable isotope labeling (SIL) has emerged as a powerful tool to measure the relative quantitative differences between samples in many differential display-type proteomic applications. However, current SIL procedures tend to suffer from the fact that one needs to decide very early in a biochemical strategy whether or not a sample will be subjected to relative quantification. Typically, the entire strategy has to be adapted to the needs of the particular quantification method chosen which might limit the range of biochemical experiments amenable to quantification. Metabolic labeling approaches, albeit very sensitive, can only be applied to studies using appropriate cell culture systems which might not necessarily be compatible with the biological system under investigation. Chemical labeling of complex protein mixtures by, e.g., isotope-coded affinity tags (ICAT), can offer great simplification of protein mixtures but is restricted by the accessibility of the often few suitable peptides (i.e. cysteine containing peptides) for both protein identification and quantification. Here, we describe a post-digest (18)O-labeling method that can circumvent some of the above limitations by separating protein identification from quantification. An aliquot of all samples in a set can be used for rapid protein ID using, e.g., matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOFMS). In a second step, relative quantification is performed using trypsin-catalyzed (18)O incorporation into all tryptic peptides. This two-stage procedure introduces significant experimental flexibility because it enables postponement of the decision about which pairs of samples from a given set of experiments are to be compared until after the protein ID stage. In-gel digested protein quantities between 50 fmol and 15 pmol are amenable to this new method, with a dynamic range of 1:10 within one sample. Accuracy for measured relative abundances is similar to those reported for other SIL strategies (errors typically <20%), and the method is applicable to protein samples from all kinds of tissue or cell culture. This paper presents quantification data for a set of standard proteins, as well as a study of differential complex formation around the NFkappaB transcription factor p65 following stimulation with TNF-alpha.
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PMID:Femtomol sensitivity post-digest (18)O labeling for relative quantification of differential protein complex composition. 1509 55

The biological role of most proteases in vivo is largely unknown. Therefore, to develop robust techniques to analyze the protease degradome in cells and tissues and to elucidate their substrate degradomes we have developed a dedicated and complete human protease and inhibitor microarray that we have called the CLIP-CHIP Oligonucleotides (70-mers) for identifying all 715 human proteases, inactive homologs and inhibitors were spotted in triplicate onto glass slides with a dedicated subarray containing oligonucleotides for specific human breast carcinoma genes. Initial analyses revealed the elevated expression of a number of proteases in invasive ductal cell carcinoma including ADAMTS17, carboxypeptidases A5 and M, tryptase-gamma and matriptase-2. Matrix metalloproteinases (MMPs) showed a restricted expression pattern in both normal and cancerous breast tissues with most expressed at low levels. However, of the several MMPs expressed in significant quantities, the carcinoma samples showed only slightly elevated amounts other than for MMP-28 which was strongly elevated. To discover new protease substrates we developed a novel yeast two-hybrid approach we term 'inactive catalytic domain capture' (ICDC). Here, an inactive mutant protease catalytic domain lacking the propeptide was used as a yeast two hybrid bait to screen a human fibroblast cDNA library for interactor proteins as a substrate trap. Wnt-induced signaling protein-2 (WISP-2) was identified by ICDC and was biochemically confirmed as a new MMP substrate. In another approach we used isotope-coded affinity tag (ICAT) labeling with tandem mass spectrometry to quantitate the levels of secreted or shed extracellular proteins in MDA-MB-231 breast carcinoma cell cultures in the presence or absence of membrane type 1-MMP (MT1-MMP) overexpression. By this proteomic approach we identified and biochemically confirmed that IL-8, the serine protease inhibitor SLPI, the death receptor-6, pro-TNF-alpha and CTGF are novel substrates of MT1-MMP. The utility and quantitative nature of ICAT with MS/MS analysis as a new screen for protease substrate discovery based on detection of cleaved or shed substrate products should be readily adaptable to other classes of protease for assessing proteolytic function in a cellular context.
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PMID:Protease degradomics: mass spectrometry discovery of protease substrates and the CLIP-CHIP, a dedicated DNA microarray of all human proteases and inhibitors. 1525 81

In acute pancreatitis, ICAM-1 is upregulated in various organs and contributes to the development of organ injury. To investigate the effects of pancreatic proteases on ICAM-1 expression and their role in the early process of leukocyte migration, human umbilical vein endothelial cells (HUVECs) were incubated with serum subjected to limited trypsin digestion and Wistar rats were injected with trypsin. Significant upregulation of membrane-bound ICAM-1 was seen on HUVECs incubated with trypsinated serum. Likewise, soluble ICAM-1 increased in the supernatant of HUVECs. Changes of membrane-bound ICAM-1 and soluble ICAM-1 were maximal with high concentrations of trypsin. HUVECs incubated with TNF-alpha (positive control) showed similar changes. In the pancreas and lungs of animals infused with trypsin, ICAM-1 and leukocyte sequestration were increased compared with controls. Reflecting the relevance of protease-induced ICAM-1 expression in leukocyte migration, leukocyte-endothelium interaction, as assessed by intravital microscopy, was markedly increased by trypsin. Inhibition of ICAM-1 ameliorated these changes significantly. In conclusion, trypsinated serum induces the upregulation of both membrane-bound ICAM-1 on endothelial cells and soluble ICAM-1. These changes contribute to the early steps of leukocyte migration in acute pancreatitis. The role of soluble ICAM-1 remains to be investigated.
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PMID:Membrane-bound ICAM-1 is upregulated by trypsin and contributes to leukocyte migration in acute pancreatitis. 1530 72

Previous studies demonstrated that live Bartonella quintana often induces angioproliferative lesions in humans. It modulates endothelial cell apoptotic and inflammatory patterns, thus inducing a very early overexpression of caspase 8 and Apaf-1 and increasing mRNA production of TNF-alpha, interleukin-8, and E-selectin. However, starting at 10 hours postinfection, the bacteria provoke antiapoptotic effects that induce an increase of bcl-2 gene transcription. To gain further insight into the cellular mechanisms that regulate apoptosis, survival and proliferation, we studied the modulation of mitogen-activated protein kinase (MAPK) and the activation state of cdc2 kinase, which regulates progression into mitosis. Confocal microscopy findings indicated a maximum rate of Bartonella entry into host cells between postinfection hours 6 and 10. Live bacteria caused substantially higher apoptosis of human umbilical vein endothelial cells-cryopreserved (HUVEC-C) than heat- and trypsin-inactivated microorganisms. During the first 6 hours postinfection, B. quintana triggered a peak of apoptosis, induced activation of p38 MAPK and stress-activated protein kinase/c-Jun N-terminal kinase (SAPK/JNK), with bacterial clusters appearing at the cellular surface of the HUVEC-C. However, at 8 to 24 hours postinfection, B. quintana was internalized and inhibited proapoptotic signals such as p38 MAPK and SAPK/JNK while inducing antiapoptotic signals. Indeed, expression of the bcl-2 gene and the increase of the bcl-2 kinase active form was concomitant to activation of mitosis, as shown by cdc2 protein activation. These data thus suggest that mechanisms that induce mitotic activity and inhibit apoptotic signals may contribute to the ability of B. quintana to cause vascular proliferation.
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PMID:Bartonella quintana-induced apoptosis inhibition of human endothelial cells is associated with p38 and SAPK/JNK modulation and with stimulation of mitosis. 1554

Pleural inflammation underlies many pleural diseases, but its pathogenesis remains unclear. Proteinase-activated receptor-2 (PAR(2)) is a novel seven-transmembrane receptor with immunoregulatory roles. We hypothesized that PAR(2) is present on mesothelial cells and can induce pleural inflammation. PAR(2) was detected by immunohistochemistry in all (19 parietal and 11 visceral) human pleural biopsies examined. In cultured murine mesothelial cells, a specific PAR(2)-activating peptide (SLIGRL-NH(2)) at 10, 100, and 1,000 muM stimulated a 3-, 42-, and 1,330-fold increase of macrophage inflammatory protein (MIP)-2 release relative to medium control, respectively (P < 0.05 all) and a 2-, 32-, and 75-fold rise over the control peptide (LSIGRL-NH(2), P < 0.05 all). A similar pattern was seen for TNF-alpha release. Known physiological activators of PAR(2), tryptase, trypsin, and coagulation factor Xa, also stimulated dose-dependent MIP-2 release from mesothelial cells in vitro. Dexamethasone inhibited the PAR(2)-mediated MIP-2 release in a dose-dependent manner. In vivo, pleural fluid MIP-2 levels in C57BL/6 mice injected intrapleurally with SLIGRL-NH(2) (10 mg/kg) were significantly higher than in mice injected with LSIGRL-NH(2) or PBS (2,710 +/- 165 vs. 880 +/- 357 vs. 88 +/- 46 pg/ml, respectively; P < 0.001). Pleural fluid neutrophil counts were higher in SLIGRL-NH(2) group than in the LSIGRL-NH(2) and PBS groups (by 40- and 26-fold, respectively; P < 0.05). This study establishes that activation of mesothelial cell PAR(2) potently induces the release of inflammatory cytokines in vitro and neutrophil recruitment into the pleural cavity in vivo.
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PMID:Activation of proteinase-activated receptor-2 in mesothelial cells induces pleural inflammation. 1559 15

Lonicera japonica Thunb.(Caprifoliaceae) has long been known as an anti-inflammatory. In the present study, the effect of water fraction of Lonicera japonica (LJ) on trypsin-induced mast cell activation was examined. HMC-1 cells were stimulated with trypsin (100 nM) in the presence or absence of LJ (10, 100, and 1000 microg/mL). TNF-alpha and tryptase production were measured by enzyme-linked immunosorbent assay (ELISA) and reverse transcription-PCR. Extracellular signal-regulated kinase (ERK) phosphorylation was assessed by Western blot. Trypsin activity was measured by using Bz-DL-Arg-p-nitroanilide (BAPNA) as substrate. LJ (10, 100, and 1000 microg/mL) inhibited TNF-alpha secretion in a dose-dependent manner. LJ (10, 100, and 1000 microg/mL) also inhibited TNF-alpha and tryptase mRNA expression in trypsin-stimulated HMC-1. Furthermore, LJ inhibited trypsin-induced ERK phosphorylation. However, LJ did not affect the trypsin activity even 1000 microg/mL. These results indicate that LJ may inhibit trypsin-induced mast cell activation through the inhibition of ERK phosphorylation than the inhibition of trypsin activity.
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PMID:Inhibition of trypsin-induced mast cell activation by water fraction of Lonicera japonica. 1559 18

Mast cells are critical for allergic reactions, but also for innate or acquired immunity and inflammatory conditions that worsen by stress. Corticotropin-releasing hormone (CRH), which activates the hypothalamic-pituitary-adrenal axis under stress, also has proinflammatory peripheral effects possibly through mast cells. We investigated the expression of CRH receptors and the effects of CRH in the human leukemic mast cell (HMC-1) line and human umbilical cord blood-derived mast cells. We detected mRNA for CRH-R1alpha, 1beta, 1c, 1e, 1f isoforms, as well as CRH-R1 protein in both cell types. CRH-R2alpha (but not R2beta or R2gamma) mRNA and protein were present only in human cord blood-derived mast cells. CRH increased cAMP and induced secretion of vascular endothelial growth factor (VEGF) without tryptase, histamine, IL-6, IL-8, or TNF-alpha release. The effects were blocked by the CRH-R1 antagonist antalarmin, but not the CRH-R2 antagonist astressin 2B. CRH-stimulated VEGF production was mediated through activation of adenylate cyclase and increased cAMP, as evidenced by the fact that the effect of CRH was mimicked by the direct adenylate cyclase activator forskolin and the cell-permeable cAMP analog 8-bromo-cAMP, whereas it was abolished by the adenylate cyclase inhibitor SQ22536. This is the first evidence that mast cells express functional CRH receptors and that CRH can induce VEGF secretion selectively. CRH-induced mast cell-derived VEGF could, therefore, be involved in chronic inflammatory conditions associated with increased VEGF, such as arthritis or psoriasis, both of which worsen by stress.
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PMID:Human mast cells express corticotropin-releasing hormone (CRH) receptors and CRH leads to selective secretion of vascular endothelial growth factor. 1594 67


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