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)

Upon entering mammalian cells, Pseudomonas exotoxin A (PE) is proteolytically processed by furin to produce an N-terminal fragment of 28 kDa and a C-terminal fragment of 37 kDa. Cleavage is followed by the reduction of a key disulfide bond (cysteines 265-287). This combination of proteolysis and reduction releases the 37 kDa C-terminal fragment, which then translocates to the cytosol where it ADP-ribosylates elongation factor 2 and inhibits protein synthesis. To investigate toxin reduction, furin-nicked PE or a hypercleavable mutant, PEW281A, was subjected to various treatments and then analyzed for fragment production. Reduction was evident only when unfolding conditions and a reducing agent were applied. Thermal unfolding of PE, as evidenced by changes in alpha-helical content and increased sensitivity to trypsin, rendered nicked toxin susceptible to protein disulfide isomerase- (PDI-) mediated reduction. When subcellular fractions from toxin-sensitive cells were incubated with nicked PE, toxin unfolding and reducing activities were present in the membrane fraction but not the soluble fraction. These data indicate that PE reduction is a two-step process: unfolding that allows access to the Cys265-287 disulfide bond, followed by reduction of the sulfur-sulfur bond by PDI or a PDI-like enzyme. With regard to cellular processing, we propose that the toxin's three-dimensional structure retains a "closed" conformation that restricts solvent access to the Cys265-287 disulfide bond until after a cell-mediated unfolding event.
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PMID:Reduction of furin-nicked Pseudomonas exotoxin A: an unfolding story. 1060 Jan 12

Immunosuppression induced by measles virus (MV) is associated with unresponsiveness of peripheral blood lymphocytes (PBL) to mitogenic stimulation ex vivo and in vitro. In mixed lymphocyte cultures and in an experimental animal model, the expression of the MV glycoproteins on the surface of UV-inactivated MV particles, MV-infected cells, or cells transfected to coexpress the MV fusion (F) and the hemagglutinin (H) proteins was found to be necessary and sufficient for this phenomenon. We now show that MV fusion-inhibitory peptides do not interfere with the induction of immunosuppression in vitro, indicating that MV F-H-mediated fusion is essentially not involved in this process. Proteolytic cleavage of MV F(0) protein by cellular proteases, such as furin, into the F(1)-F(2) subunits is, however, an absolute requirement, since (i) the inhibitory activity of MV-infected BJAB cells was significantly impaired in the presence of a furin-inhibitory peptide and (ii) cells expressing or viruses containing uncleaved F(0) proteins revealed a strongly reduced inhibitory activity which was improved following trypsin treatment. The low inhibitory activity of effector structures containing mainly F(0) proteins was not due to an impaired F(0)-H interaction, since both surface expression and cocapping efficiencies were similar to those found with the authentic MV F and H proteins. These results indicate that the fusogenic activity of the MV F-H complexes can be uncoupled from their immunosuppressive activity and that the immunosuppressive domains of these proteins are exposed only after proteolytic activation of the MV F(0) protein.
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PMID:Proteolytic cleavage of the fusion protein but not membrane fusion is required for measles virus-induced immunosuppression in vitro. 1064 71

Proteolytic cleavage of the fusion protein (F) is an important control mechanism of the biological activity of paramyxoviruses. The sequence R-R-H-K-R(112) at the cleavage site of the F protein of measles virus (MV) was altered by site-directed mutagenesis to R-N-H-N-R(112), which is not recognized by the ubiquitous cellular protease furin. When transiently expressed in cell cultures standard F protein was cleaved, whereas the mutant remained in the uncleaved form. Syncytium formation by the mutant that was analysed after coexpression with haemagglutinin protein depended on the presence of trypsin. Recombinant MV containing the mutation required trypsin activation for fusion and infectivity in cell culture. Intranasal infection of transgenic mice susceptible to MV infection (Ifnar(tm)-CD46Ge) resulted in a moderately productive infection and inflammation of the lung. In contrast to parental virus, intracerebral inoculation did not induce neural disease. The possible effects of the change in cleavage activation on tissue tropism and pathogenicity are discussed.
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PMID:Recombinant measles virus requiring an exogenous protease for activation of infectivity. 1064 43

The F (fusion) protein of the respiratory syncytial viruses is synthesized as an inactive precursor F(0) that is proteolytically processed at the multibasic sequence KKRKRR(136) into the subunits F(1) and F(2) by the cellular protease furin. This maturation process is essential for the F protein to gain fusion competence. We observed that proteolytic cleavage additionally occurs at another basic motif, RARR(109), that also meets the requirements for furin recognition. Cleavage at both sites leads to the removal from the polypeptide chain of a glycosylated peptide of 27 amino acids. When the sequence RARR(109) was changed to NANR(109) or to RANN(109) by site-directed mutagenesis, cleavage by furin was completely prevented. Although the mutants were still processed at position Arg(136), they did not show any syncytia formation. Proteolytic cleavage of the modified motifs was achieved by treatment of transfected cells with trypsin converting the F mutants into their fusogenic forms. Our findings indicate that both furin consensus sequences have to be cleaved in order to activate the fusion protein.
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PMID:Proteolytic activation of respiratory syncytial virus fusion protein. Cleavage at two furin consensus sequences. 1141 98

We have examined the consequences of cleaving the fusion glycoprotein (F) of human respiratory syncytial virus (HRSV) at two distinct furin-recognition sites. Purified anchorless F is a mixture of unaggregated cone-shaped molecules and rosettes of lollipop-shaped spikes. The unaggregated molecules contain a proportion of uncleaved F0 and an intermediate, F(delta1-109), cleaved only at site I, residues 106-109. Inhibition of cleavage at site I, by two amino acid changes (R108N/R109N), reduces the proportion of aggregated molecules with a concomitant increase in the amount of unprocessed F0. Inhibition of cleavage at site II, residues 131-136, by deletion of four amino acids (delta131-134), abrogates aggregation of anchorless F and all molecules are seen as individual cone-shaped rods. In vitro cleavage of anchorless F, or mutant delta131-134, with trypsin at 4, 20, or 37 degrees C, under conditions in which cleavage at site II is complete in all molecules, leads to their aggregation in rosettes of lollipop-shaped spikes. Thus, cleavage at site II is required for the structural changes in anchorless F that lead to changes in shape and to aggregation. The segment between sites I and II, residues 110-136, is not associated with anchorless F in the supernatant of infected cell cultures, indicating that it is released from the processed protein when cleavage at sites I and II is completed.
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PMID:Effect of proteolytic processing at two distinct sites on shape and aggregation of an anchorless fusion protein of human respiratory syncytial virus and fate of the intervening segment. 1212 93

This study demonstrates that endopin 2 is a unique secretory vesicle serpin that displays cross-class inhibition of cysteine and serine proteases, indicated by effective inhibition of papain and elastase, respectively. Homology of the reactive site loop (RSL) domain of endopin 2, notably at P1-P1' residues, with other serpins that inhibit cysteine and serine proteases predicted that endopin 2 may inhibit similar proteases. Recombinant N-His-tagged endopin 2 inhibited papain and elastase with second-order rate constants (k(ass)) of 1.4 x 10(6) and 1.7 x 10(5) M(-1) s(-1), respectively. Endopin 2 formed SDS-stable complexes with papain and elastase, a characteristic property of serpins. Interactions of the RSL domain of endopin 2 with papain and elastase were indicated by cleavage of endopin 2 near the predicted P1-P1' residues by these proteases. Endopin 2 did not inhibit the cysteine protease cathepsin B, or the serine proteases chymotrypsin, trypsin, plasmin, and furin. Endopin 2 in neuroendocrine chromaffin cells was colocalized with the secretory vesicle component (Met)enkephalin by confocal immunonfluorescence microscopy, and was present in isolated secretory vesicles (chromaffin granules) from chromaffin cells as a glycoprotein of 72-73 kDa. Moreover, regulated secretion of endopin 2 from chromaffin cells was induced by nicotine and KCl depolarization. Overall, these results demonstrate that the serpin endopin 2 possesses dual specificity for inhibiting both papain-like cysteine and elastase-like serine proteases. These findings demonstrate that endopin 2 inhibitory functions may occur in the regulated secretory pathway.
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PMID:The novel serpin endopin 2 demonstrates cross-class inhibition of papain and elastase: localization of endopin 2 to regulated secretory vesicles of neuroendocrine chromaffin cells. 1217 26

Proteolytic processing of the respiratory syncytial virus F (fusion) protein results in the generation of the disulfide-linked subunits F1 and F2 and in the release of pep27, a glycopeptide originally located between the two furin cleavage sites FCS-1 (RKRR(136)) and FCS-2 (RAR/KR(109)). We made use of reverse genetics to study the importance of FCS-2 and of pep27 for BRSV replication in cell culture. Replacement of FCS-2 in the F protein of recombinant viruses by either of the sequences NANR(109), RANN(109) or SANN(109), respectively, abolished proteolytic processing at this position, whereas the cleavage of FCS-1 was not affected. All mutants replicated in calf kidney and Vero cells in the absence of exogenous trypsin, although somewhat higher titers of BRSV containing the NANR(109) or the RANN(109) motif were achieved in the presence of trypsin. The virus mutants showed a reduced cytopathic effect which was lowest in the case of the SANN(109) mutant. These findings demonstrate that cleavage at FCS-2 is dispensable for replication of respiratory syncytial virus in cell culture. A deletion mutant containing FCS-1 but lacking FCS-2 and most of pep27 replicated in cell culture as efficiently as the parental virus, indicating that this domain of the F protein is not essential for virus maturation and infectivity.
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PMID:Cleavage at the furin consensus sequence RAR/KR(109) and presence of the intervening peptide of the respiratory syncytial virus fusion protein are dispensable for virus replication in cell culture. 1218 5

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

Secretory vesicles of neuroendocrine cells possess multiple proteases for proteolytic processing of proteins into biologically active peptide components, such as peptide hormones and neurotransmitters. The importance of proteases within secretory vesicles predicts the presence of endogenous protease inhibitors in this subcellular compartment. Notably, serpins represent a diverse class of endogenous protease inhibitors that possess selective target protease specificities, defined by the reactive site loop domains (RSL). In the search for endogenous serpins in model secretory vesicles of neuroendocrine chromaffin cells, the presence of serpins related to alpha1-antichymotrypsin (ACT) was detected by Western blots with anti-ACT. Molecular cloning revealed the primary structures of two unique serpins, endopin 1 and endopin 2, that possess homology to ACT. Of particular interest was the observation that distinct RSL domains of these new serpins predicted that endopin 1 would inhibit trypsin-like serine proteases cleaving at basic residues, and endopin 2 would inhibit both elastase and papain that represent serine and cysteine proteases, respectively. Endopin 1 showed selective inhibition of trypsin, but did not inhibit chymotrypsin, elastase, or subtilisin. Endopin 2 demonstrated cross-class inhibition of the cysteine protease papain and the serine protease elastase. Endopin 2 did not inhibit chymotrypsin, trypsin, plasmin, thrombin, furin, or cathepsin B. Endopin 1 and endopin 2 each formed SDS-stable complexes with target proteases, a characteristic property of serpins. In neuroendocrine chromaffin cells from adrenal medulla, endopin 1 and endopin 2 were both localized to secretory vesicles. Moreover, the inhibitory activity of endopin 2 was optimized under reducing conditions, which required reduced Cys-374; this property is consistent with the presence of endogenous reducing agents in secretory vesicles in vivo. These new findings demonstrate the presence of unique secretory vesicle serpins, endopin 1 and endopin 2, which possess distinct target protease selectivities. Endopin 1 inhibits trypsin-like proteases; endopin 2 possesses cross-class inhibition for inhibition of papain-like cysteine proteases and elastase-like serine proteases. It will be of interest in future studies to define the endogenous protease targets of these two novel secretory vesicle serpins.
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PMID:Novel secretory vesicle serpins, endopin 1 and endopin 2: endogenous protease inhibitors with distinct target protease specificities. 1243 89

Flavivirus particles are synthesized in an immature form containing heterodimers of the proteins prM and E. Shortly before release from the cell, prM is cleaved by the host protease furin to yield mature virions. In this study, the furin-mediated cleavage of the tick-borne encephalitis (TBE) virus protein prM was prevented by specific mutagenesis of the cleavage site. This resulted in the production of immature TBE virions, which were shown to be completely non-infectious in BHK-21 cells. This finding contrasted with previous studies in which immature flavivirus particles produced by other techniques were shown to have considerable residual infectivity. The structural integrity of the mutant virus particles was confirmed by the characterization of physical and antigenic properties. Most importantly, infectivity could be restored by the addition of trypsin, which presumably cleaved protein prM at one of the monobasic sites retained in the mutated sequence. In the presence of trypsin, the mutant could be passaged repeatedly in BHK-21 cells, but if the protease was removed, the activated particles could initiate only a single round of infection, which again generated non-infectious virus progeny. These observations provide evidence that the infectivity of flaviviruses depends on the endoproteolytic cleavage of protein prM, which probably has a regulatory function rather than a direct role in virus entry. Moreover, the results illustrate that mutation of the furin cleavage site is a convenient way to produce single-round infectious flavivirus particles, which may be useful in vaccine and vector development.
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PMID:Cleavage of protein prM is necessary for infection of BHK-21 cells by tick-borne encephalitis virus. 1253 15


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