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:3.4.25.1 (proteasome)
28,817 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

Human immunodeficiency virus type-1 (HIV-1) reverse transcriptase (RT) plays a central role in the virus replication cycle. We found that HIV-1 RT was rapidly degraded when incubated with cell extracts obtained from human peripheral blood cells. The proteolytic activity responsible for the in vitro degradation of RT was present in monocytes and their precursors. Interestingly, this activity was downregulated upon cell activation or differentiation along the macrophage pathway. The proteolytic process appears specific for HIV-1 RT since other HIV-1 proteins were not degraded upon incubation in the same extracts. Although the degradation of RT was unaffected by specific proteasome inhibitors, it could be inhibited by PMSF and aprotinin, suggesting the involvement of a serine protease. Upon cell fractionation, this serine protease was found to be associated with the microsomal fraction and displayed an apparent molecular weight of approximately 2000 kDa, as determined by gel filtration. Our results suggest that a giant serine protease, different from tripeptidyl peptidase II, is involved in the in vitro degradation of HIV-1 RT. The possibility of an in vivo interaction between HIV-1 RT and a cell-type-specific serine protease is discussed.
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PMID:Human monocytes possess a serine protease activity capable of degrading HIV-1 reverse transcriptase in vitro. 1146 30

The thermal stability of the alkaline protease extracellular subtilisin-type serine protease (AprP) from Pseudomonas sp. KFCC 10818 was improved by altering an amino acid residue at an autoproteolytic cleavage site. N-terminal sequence analysis of the autoproteolytic products of the protein revealed the presence of two cleavage sites, Ser-307 and Ser-331. To increase the thermal stability of the enzyme, serine residues of these sites were replaced with aspartate. The S331D mutant enzyme was successfully purified and characterized whereas the S307D mutant was not. The half-lives of the S331D mutant at 55 degrees C and 60 degrees C were 1.5 and 2.4 times longer than that of the wild-type enzyme, respectively. In addition, the catalytic efficiency was also enhanced.
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PMID:Enhanced thermal stability of an alkaline protease, AprP, isolated from a Pseudomonas sp. by mutation at an autoproteolysis site, Ser-331. 1159 12

The goal of the current study was to examine the role of the ubiquitin-proteasome system (UPS), a pathway of intracellular degradation, in the regulation of fetal fibronectin (FFN) expression in human placenta. Primary cultures of cytotrophoblasts (CTs) and placental mesenchymal cells (PMCs) were isolated from human term placentas and were maintained in serum-free medium (SFM) in the presence of inhibitors of proteasome-mediated degradation (e.g., MG132) as well as inhibitors of other proteases. Levels of secreted FFN and interleukin (IL)-8 in culture media were quantitated by enzyme-linked immunosorbent assay (ELISA), and cell viability was assessed by trypan blue exclusion. Intracellular levels of FFN and ubiquinated proteins were measured by Western blotting, and levels of fibronectin mRNA were determined following Northern blotting. We found that proteasome inhibitors (MG132, MG262, and PSI) potently suppressed levels of secreted FFN in cultures of CTs, but they not did affect levels of IL-8. Lysosomal, calpain, and serine protease inhibitors as well as the anti-inflammatory compound sulfasalazine did not markedly affect levels of secreted FFN in CT cultures. Proteasome inhibitors did not compromise cell viability during the initial 16-18 hours of treatment and did not affect intracellular levels of FFN protein or fibronectin mRNA. The efficacy of suppression of FFN in CT culture media by proteasome inhibitors reflected their effects on intracellular accumulation of ubiquinated proteins. By contrast, the presence of proteasome inhibitors did not alter levels of secreted FFN in cultures of PMCs. We conclude that inhibitors of proteasome-mediated degradation potently and specifically suppressed extracellular expression of FFN in CTs through a cell type-specific pathway that did not involve alterations in FFN synthesis. This suggests that accumulation of ubiquinated proteins in the presence of proteasome inhibitors blocks FFN secretion or promotes the extracellular degradation of FFN. This experimental paradigm will be useful for dissecting the role of the UPS in regulating CT function.
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PMID:Role of the proteasome in the regulation of fetal fibronectin secretion in human placenta. 1159 54

A new engineering strain, Bacillus pumilus c172-14 (pBX 96), was obtained by introducing the pBX 96 plasmid, which carries the alpha-amylase amy gene, into the host strain of alkalophilic Bacillus pumilus c172 via transformation. The newly constructed strain was found to express the amy gene and could use starch instead of glucose or starch hydrolysate as carbon source for its fermentation of alkaline protease. The pBX 96 plasmid in the new host was found to be segregationally and structurally stable. The expression of amy gene did not affect the host strain's resistance to bacteriophages. Moreover, the level of alkaline protease was improved significantly compared with the parent strain. The constructed strain gave a maximum alkaline protease activity of 14,014 U/ml in shaking flask after 48 h cultivation when growing in a medium containing 6% corn meal, 4% soybean flour, 0.4% Na2HPO4, 0.03% KH2PO4, 0.02% MgCl2, 0.3% CaCl2, 0.25% Na2CO3, 0.1% glucose, and 20 microg/ml kanamycin (pH 7.0). The optimal pH value and temperature of the alkaline protease were 11.0 and 40 degrees C, respectively. This enzyme was stable over a pH range of 8-11. Its residual activity remained at 100% when treated under a temperature of less than 45 degrees C for 30 min. The corresponding residual activity reduced to 65% of its optimal value at 60 degrees C for 30 min. The alkaline protease was a kind of serine protease, which was demonstrated by the complete inactivation by PMSF (1 mM). This newly constructed strain will be useful in the alkaline protease industry.
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PMID:Fermentation of starch for enhanced alkaline protease production by constructing an alkalophilic Bacillus pumilus strain. 1169 13

Epithelial cells are the first cells that encounter infecting bacteria and, as such, they have developed several mechanisms for microbial protection. We have shown previously that bladder epithelial cells express the lipopolysaccharide (LPS) receptor Toll-like receptor (TLR) 4 that enables a rapid cellular interleukin (IL)-8 response when exposed to Escherichia coli and LPS. TLR4 belongs to a family of receptors that was initially identified in Drosophila, in which Toll is required for the immune response against fungi. Fungal exposure activates a series of serine proteases that process the protein Spaetzle to a cytokine-like form that acts as a ligand for Toll. Here, we investigated whether a similar proteolytic cascade is required for human TLR activation. When screening a set of 18 protease inhibitors, three serine protease inhibitors (TPCK, TLCK and Pefabloc) were shown to inhibit LPS- and peptidoglycan-induced IL-8 production in TLR2- and TLR4-positive bladder epithelial cells. However, they were equally effective inhibitors of IL-1beta-induced signalling, indicating that their target(s) is/are located downstream of the TLRs. Further characterization showed that these inhibitors blocked I kappa B degradation but not phosphorylation in LPS-stimulated cells, which suggests that the serine protease inhibitors target the 26S proteasome. Identical results were obtained on LPS-stimulated monocytes. Based on these data, we find no evidence for the involvement of proteases upstream of TLRs in either epithelial cells or cells of the monocytic lineage.
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PMID:TLR4-dependent lipopolysaccharide signalling in epithelial cells is independent of extracellular protease activity. 1202 57

C4b and C3b deposited on host cells undergo limited proteolytic cleavage by regulatory proteins. Membrane cofactor protein (MCP; CD46), factor H, and C4b binding protein mediate this reaction, known as cofactor activity, that also requires the plasma serine protease factor I. To explore the roles of the fluid phase regulators vs those expressed on host cells, a model system was used examining complement fragments deposited on cells transfected with human MCP as assessed by FACS and Western blotting. Following incubation with Ab and complement on MCP(+) cells, C4b was progressively cleaved over the first hour to C4d and C4c. There was no detectable cleavage of C4b on MCP(-) cells, indicating that MCP (and not C4BP in the serum) primarily mediates this cofactor activity. C3b deposition was not blocked on MCP(+) cells because classical pathway activation occurred before substantial C4b cleavage. Cleavage, though, of deposited C3b was rapid (<5 min) and iC3b was the dominant fragment on MCP(-) and MCP(+) cells. Studies using a function-blocking mAb further established factor H as the responsible cofactor. If the level of Ab sensitization was reduced 8-fold or if Mg(2+)-EGTA was used to block the classical pathway, MCP efficiently inhibited C3b deposition mediated by the alternative pathway. Thus, for the classical pathway, MCP is the cofactor for C4b cleavage and factor H for C3b cleavage. However, if the alternative pathway mediates C3b deposition, then MCP's cofactor activity is sufficient to restrict complement activation.
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PMID:Role of membrane cofactor protein (CD46) in regulation of C4b and C3b deposited on cells. 1205 45

An alkaline protease was isolated from culture filtrate of B. subtilis NCIM 2713 by ammonium sulphate precipitation and was purified by gel filtration. With casein as a substrate, the proteolytic activity of the purified protease was found to be optimal at pH 8.0 and temperature 70 degrees C. The purified protease had molecular weight 20 kDa, Isoelectric point 5.2 and km 2.5 mg ml(-1). The enzyme was stable over the pH range 6.5-9.0 at 37 degrees C for 3 hr. During chromatographic separation this protease was found to be susceptible to autolytic degradation in the absence of Ca2+. Ca2+ was not only required for the enzyme activity but also for the stability of the enzyme above 50 degrees C. About 62% activity was retained after 60 min at pH 8.0 and 55 degrees C. DFP and PMSF completely inhibited the activity of this enzyme, while in the presence of EDTA only 33% activity remained. However, it was not affected either by sulfhydryl reagent, or by divalent metal cations, except SDS and Hg2+. The results indicated that this is a serine protease.
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PMID:A study of extracellular alkaline protease from Bacillus subtilis NCIM 2713. 1256 22

An extracellular alkaline serine protease (called DHAP), produced by a Bacillus pumilus strain, demonstrates significant dehairing function. This protease is purified by hydrophobic interaction chromatography, ion exchange, and gel filtration. DHAP had a pI of 9.0 and a molecular weight of approximately 32,000 Dalton. It shows maximal activity at pH 10 and with a temperature of 55 degrees C; the enzyme activity can be completely inhibited by phenylmethylsulfonyl fluoride (PMSF) and diisopropyl fluorophosphates (DFP). The first 20 amino acid residues of the purified DHAP have been determined with a sequence of AQTVPYGIPQIKAPAVHAQG. Alignment of this sequence with other alkaline protease demonstrates its high homology with protease from another B. pumilus strain.
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PMID:Purification and characterization of an extracellular alkaline serine protease with dehairing function from Bacillus pumilus. 1256 38

Proteolytic activation of hemagglutinin, an envelope glycoprotein of the influenza virus, by host proteases is essential for infection and proliferation of the virus. However, there is no well-defined, inherent source of host proteases in man or swine, both of which are natural hosts for human influenza viruses. We have recently isolated a 32 kDa protein in a high salt extract from porcine lungs, which possess the hemagglutinin processing activity. In this study, we attempted to purify another hemagglutinin processing enzyme from porcine lung. The purified enzyme, named tryptase TC30, exhibited a molecular mass of about 30 kDa by SDS-PAGE and 28.5 kDa by gel filtration chromatography, suggesting that it is a monomer. Tryptase TC30 cleaved peptide substrates with Arg at the P1 position, and preferentially substrates with the Ser-Ile-Gin-Ser-Arg sequence corresponding to the HA cleavage site sequence of the A/PR/8/34 influenza virus. Among various inhibitors tested, trypsin-type serine protease inhibitors, such as aprotinin, antipain, benzamidine and leupeptin, efficiently inhibited the proteolytic activity of the enzyme. The N-terminal 40 amino acid sequence of tryptase TC30 exhibits more than 60% homology to mast cell tryptases from mice MCP-6 and human tryptase-alpha and -beta. These data indicate that tryptase TC30, the 30 kDa enzyme from porcine lung, is a novel hemagglutinin-cleaving enzyme.
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PMID:A novel influenza A virus activating enzyme from porcine lung: purification and characterization. 1267 14

CodWX in Bacillus subtilis is an ATP-dependent, N-terminal serine protease, consisting of CodW peptidase and CodX ATPase. Here we show that CodWX is an alkaline protease and has a distinct molecular architecture. ATP hydrolysis is required for the formation of the CodWX complex and thus for its proteolytic function. Remarkably, CodX has a 'spool-like' structure that is formed by interaction of the intermediate domains of two hexameric or heptameric rings. In the CodWX complex, CodW consisting of two stacked hexameric rings (WW) binds to either or both ends of a CodX double ring (XX), forming asymmetric (WWXX) or symmetric cylindrical particles (WWXXWW). CodWX can also form an elongated particle, in which an additional CodX double ring is bound to the symmetric particle (WWXXWWXX). In addition, CodWX is capable of degrading EzrA, an inhibitor of FtsZ ring formation, implicating it in the regulation of cell division. Thus, CodWX appears to constitute a new type of protease that is distinct from other ATP-dependent proteases in its structure and proteolytic mechanism.
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PMID:Molecular architecture of the ATP-dependent CodWX protease having an N-terminal serine active site. 1280 5


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