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)

Flavivirus genomic RNA is translated into a large polyprotein that is processed into structural and nonstructural proteins. The N-termini of several nonstructural proteins are produced by cleavage at dibasic sites by a two-component viral proteinase consisting of NS2B and NS3. NS3 contains a trypsin-like serine proteinase domain at its N-terminus, whereas the function of NS2B in proteolysis is yet to be determined. We have used an NS3-specific antiserum, under nondenaturing conditions, to demonstrate that NS2B and NS3 form a complex both in vitro and in vivo. The N-terminal 184 residues of NS3 are sufficient to form the complex with NS2B. The complex forms efficiently when the NS2B and NS3 are translated from two different mRNAs as well as when NS2B and NS3 are translated as a polyprotein from the same mRNA. A chimeric complex can be formed between yellow fever NS2B and a chimeric yellow fever-dengue 2 NS3. Using anti-NS3 antisera, we also found that a 50-kDa fragment of NS3, consisting of the N-terminal approximately 460 residues, is produced in infected mammalian cells. This fragment is not produced in infected mosquito cells, but will form in Triton X-100 lysates of mosquito cells. The cleavage of NS3 to form this fragment is catalyzed by the NS3 proteinase itself and proteolysis requires NS2B. Examination of the amino acid sequence of NS3 reveals a potential conserved cleavage site that resembles other sites cleaved by the NS3/NS2B proteinase; this site occurs within a conserved RNA helicase sequence motif. The importance of this alternatively processed form of NS3 and its role in the replication cycle of dengue virus remain to be determined.
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PMID:Dengue 2 virus NS2B and NS3 form a stable complex that can cleave NS3 within the helicase domain. 846 Apr 92

For comparison of the physiological potential of single thyroid cells versus cells integrated into follicles it would be ideal to work with suspensions consisting exclusively of single cells instead of a mixture of single cells and follicle fragments. In this study, various techniques for the isolation of single cells have been tested for their effect on cell viability, the ultrastructure of the isolated cells, the percentage of single cells and the ability of these cells to form follicles in culture. In addition, the cells were characterized for the preservation of their morphology and the ability to respond to TSH by comparing their immunocytochemical staining pattern with anti-vimentin and anti-ras p21 antibody to that of the intact thyroid tissue. Dispase treatment of thyroid tissues alone produced suspensions with a relatively small proportion of single cells. These cells stained with anti-vimentin and anti-ras p21 antibody to a similar percentage as thyroid cells in the intact gland. A combination of dispase treatment with either filtration or trypsin treatment severely compromised the viability of the cells. A high proportion of single cells with a good viability could be obtained either by centrifugation of dispase treated tissues or by culturing of dispase treated tissues as monolayers and subsequent detachment from the culture vessels with trypsin. Whereas the immunological staining with anti-vimentin and anti-ras oncogene antibody in the centrifuged cells resembled that of intact tissue, cells cultured as monolayers reacted differently. The differences in the immunological staining were still observed when the cells which had been grown as monolayers were stimulated with TSH. Differential centrifugation appeared to be the ideal method for the isolation of unaltered and viable single cells but is a rather laborious method to obtain larger amounts of single thyroid cells.
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PMID:Efficiency of various dissociation methods for the preparation of thyroid single cell suspensions. 853 60

Plasminogen activation is observed in the human epidermis during reepithelialization of epidermal defects and under certain pathological conditions. The activation reaction depends on keratinocyte-associated plasminogen activators (PAs), which convert the ubiquitous proenzyme plasminogen into the active trypsin-like serine proteinase plasmin. The PAs are controlled by PA inhibitors (PAIs), of which two major types are known: PAI-1 and PAI-2. In vitro and in vivo keratinocytes express both PAIs. In the current study, we have addressed the possible function of PAI-2 in regulating extracellular PA activity in cultured normal human epidermal keratinocytes (NHEK), the human keratinocyte cell line (HaCaT), and a Ha-ras transfected HaCaT variant (HaRas). PAI-2 was detected intracellularly in all three cell types. Whereas only the NHEK and the HaCaT cells secreted detectable levels of PAI-2 into the culture medium, all three cell types released urokinase-type PA (uPA) into the supernatants. When comparing HaCaT and HaRas cells, we found that the cell lines secreted comparable levels of uPA antigen, whereas the levels of uPA activity were low in the presence of PAI-2, indicating that PAI-2 serves to regulate uPA activity. This assumption was supported by the findings that PAI-2 formed complexes with secreted uPA and that uPA/PAI-2 complexes were present at the surface of the PAI-2-secreting HaCaT cells but not at the surface of PAI-2 nonsecreting HaRas cells. Finally, PAI-2 was found to counteract the uPA-dependent and plasmin-mediated detachment of cultured HaCaT cells. Taken together, our findings indicate that secreted PAI-2 serves to regulate the activity of extracellular uPA in keratinocytes.
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PMID:Plasminogen activator inhibitor type-2 (PAI-2) in human keratinocytes regulates pericellular urokinase-type plasminogen activator. 863

ONO-3403 and ONO-5046 are potent synthetic protease inhibitors of trypsin and elastase, respectively. These compounds suppressed the proliferation of polyoma virus- and Kirsten sarcoma virus-transformed BALB/c 3T3 cells more effectively than their normal counterparts. SV40-transformed 3Y1 and v-Ha-ras-transformed NIH3T3 cells were also more sensitive to ONO-3403 and ONO-5046 than the parent normal cells. These results suggest that ONO-3403 and ONO-5046 are useful for selective suppression of the proliferation of rapidly growing transformed cells.
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PMID:Growth-suppressive activities of serine protease inhibitors, ONO-3403 and ONO-5046, toward normal and transformed murine fibroblasts. 871 7

During replication of hepatitis C virus (HCV), the final steps of polyprotein processing are performed by a viral proteinase located in the N-terminal one-third of nonstructural protein 3. The structure of NS3 proteinase from HCV BK strain was determined by X-ray crystallography at 2.4 angstrom resolution. NS3P folds as a trypsin-like proteinase with two beta barrels and a catalytic triad of His-57, Asp-81, Ser-139. The structure has a substrate-binding site consistent with the cleavage specificity of the enzyme. Novel features include a structural zinc-binding site and a long N-terminus that interacts with neighboring molecules by binding to a hydrophobic surface patch.
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PMID:The crystal structure of hepatitis C virus NS3 proteinase reveals a trypsin-like fold and a structural zinc binding site. 886 16

The platelet-activating factor PAF (1-O-alkyl-2-acetyl-sn-glycero-3-phosphocholine) is a potent lipid first messenger active in general cell activation, fertilization, inflammatory and allergic reactions, asthma, HIV pathogenesis, carcinogenesis, and apoptosis. There is substantial evidence that PAF is involved in intracellular signalling, but the pathways are poorly understood. Inactivation of PAF is carried out by specific intra- and extracellular acetylhydrolases (PAF-AHs), a subfamily of phospholipases A2 that remove the sn-2 acetyl group. Mammalian brain contains at least three intracellular isoforms, of which PAF-AH(Ib) is the best characterized. This isoform contains a heterodimer of two homologous catalytic subunits alpha1 and alpha2, each of relative molecular mass 26K, and a non-catalytic 45K beta-subunit, a homologue of the beta-subunit of trimeric G proteins. We now report the crystal structure of the bovine alpha1 subunit of PAF-AH(Ib) at 1.7 A resolution in complex with a reaction product, acetate. The tertiary fold of this protein is closely reminiscent of that found in p21(ras) and other GTPases. The active site is made up of a trypsin-like triad of Ser 47, His 195 and Asp 192. Thus, the intact PAF-AH(Ib) molecule is an unusual G-protein-like (alpha1/alpha2)beta trimer.
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PMID:Brain acetylhydrolase that inactivates platelet-activating factor is a G-protein-like trimer. 898 54

A cell line of mesodermal origin, FS9, was found to release a Cell Orienting Factor into its culture medium. In contrast with the random migration of controls, the orienting activity causes migrating mesenchymal cells to form an orderly "halo' surrounding tissue explants; individual cells and their cytoskeletons are elongated and parallel to each other but at right angle to the explant. No effect on the rate of cell movement was apparent. The orienting activity could be quantified by counting the number of cells found within strings radiating at right angles to a single tissue explant in the presence of FS9 conditioned medium or by using NIH image analysis. A dose dependent relationship with half maximal activity occurring at a 25% dilution of conditioned medium was observed. Cells that migrated randomly in the absence of conditioned medium became oriented within 4 h of exposure to 50% conditioned medium. Conversely, when the conditioned medium was removed, parallel alignment was rapidly lost. The orienting activity was found in conditioned media from a variety of mesodermal derivatives. Transformation of Balb/c 3T3 cells using EJ-ras oncogene led to augmented production of the activity. Furthermore, insulin was required in serum-free medium to support its production, Laminin, fibronectin and collagen and a range of pure cytokines, neither promoted nor inhibited orientation. Cell alignment was also unaffected by treatments which interfered with cell-substrate interactions and motility including the addition of the RGD peptide or anti-integrin beta 1 and beta 3 antibodies. A protein is likely to be involved since the activity was heat and trypsin sensitive and non-dialysable. The possibility is discussed that the orienting activity is a novel protein(s) which alters intercellular interactions to promote the formation of an aligned pattern by migrating mesenchymal cells.
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PMID:Detection and characterization of an activity which aligns mesodermal cells into parallel arrays. 912 62

This study evaluated the inhibitory effects of thiazolidine derivatives on hepatitis C virus (HCV) protease and other human serine proteases. The inhibition efficacy was tested with a reversed-phase high-performance liquid chromatography (HPLC) assay system using a NS3-NS4A fusion protein as the HCV protease and a synthetic peptide substrate that mimics the NS5A-5B junction. Nine thiazolidine derivatives showed more than 50% inhibition at 50 microg/ml. The most potent derivative was RD4-6250, with 50% inhibition at a concentration of 2.3 microg/ml; this concentration was lower than those of other protease inhibitors reported previously. The most selective derivative was RD4-6205, with 50% inhibition at a concentration of 6.4 microg/ml, a lower concentration than those on other serine proteases (chymotrypsin, trypsin, plasmin, and elastase). These results suggest that the RD4-6205 skeleton is an important structure for inhibitory activity on the HCV protease NS3-NS4A.
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PMID:Novel hepatitis C virus protease inhibitors: thiazolidine derivatives. 929 67

The flavivirus protease is composed of two viral proteins, NS2B and NS3. The amino-terminal portion of NS3 contains sequence and structural motifs characteristic of bacterial and cellular trypsin-like proteases. We have undertaken a mutational analysis of the region of NS3 which contains the catalytic serine, five putative substrate binding residues, and several residues that are highly conserved among flavivirus proteases and among all serine proteases. In all, 46 single-amino-acid substitutions were created in a cloned NS2B-NS3 cDNA fragment of dengue virus type 2, and the effect of each mutation on the extent of self-cleavage of the NS2B-NS3 precursor at the NS2B-NS3 junction was assayed in vivo. Twelve mutations almost completely or completely inhibited protease activity, 9 significantly reduced it, 14 decreased cleavage, and 11 yielded wild-type levels of activity. Substitution of alanine at ultraconserved residues abolished NS3 protease activity. Cleavage was also inhibited by substituting some residues that are conserved among flavivirus NS3 proteins. Two (Y150 and G153) of the five putative substrate binding residues could not be replaced by alanine, and only Y150 and N152 could be replaced by a conservative change. The two other putative substrate binding residues, D129 and F130, were more freely substitutable. By analogy with the trypsin model, it was proposed that D129 is located at the bottom of the substrate binding pocket so as to directly interact with the basic amino acid at the substrate cleavage site. Interestingly, we found that significant cleavage activity was displayed by mutants in which D129 was replaced by E, S, or A and that low but detectable protease activity was exhibited by mutants in which D129 was replaced by K, R, or L. Contrary to the proposed model, these results indicate that D129 is not a major determinant of substrate binding and that its interaction with the substrate, if it occurs at all, is not essential. This mutagenesis study provided us with an array of mutations that alter the cleavage efficiency of the dengue virus protease. Mutations that decrease protease activity without abolishing it are candidates for introduction into the dengue virus infectious full-length cDNA clone with the aim of creating potentially attenuated virus stocks.
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PMID:Mutagenesis of the NS3 protease of dengue virus type 2. 942 Feb 67

Dengue viruses are members of the Flaviviridae and cause dengue fever and the more severe dengue hemorrhagic fever. Although nearly 40 % of the world's population is at risk of dengue infection, there is currently no effective vaccine or chemotherapy for the disease. Processing of the dengue polyprotein into structural and non-structural proteins in a host, which is essential for assembly of infective virions, is carried out by the combined action of host proteases and the trypsin-like, two-component viral NS2B/NS3 serine protease. Although NS2B strongly stimulates the catalytic NS3 protease domain, the latter is fully active against small substrates and possesses detectable activity against larger substrates, making both forms of the enzyme possible targets for drug design. In the crystal structure of a complex of the protease with a Bowman-Birk inhibitor reported here, an Arg residue at the P1 position of the inhibitor is bound in a manner distinctly different from that in other serine proteases of comparable specificity. However, because the regulatory component, NS2B, is not present in the complex, the physiological implications of this observations are currently unclear. The redundant nature of interaction of P1 Arg and Lys residues with Asp129, Tyr150 and Ser163 of the enzyme provides an explanation for the observed behavior of several site-specific mutants of Asp129 in the protease. The strong level of conservation of residues in the protease that interact with the P1 Arg, along with conservation of Arg at P1 of most cleavage sites in other flaviviruses, suggests that observations from this structure are likely to be applicable to many flaviviruses. The structure provides a starting point for design of site-specific mutations to probe the mechanism of catalysis by the catalytic domain, its activation by the regulatory domain and for design of specific inhibitors of enzymatic activity.
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PMID:Crystal structure of Dengue virus NS3 protease in complex with a Bowman-Birk inhibitor: implications for flaviviral polyprotein processing and drug design. 2035 76


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