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:3.4.21.1 (chymotrypsin)
10,938 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

We studied the role of proteases in apoptosis using a cell-free system prepared from a human leukemia cell line. HL60 cells are p53 null and extremely sensitive to a variety of apoptotic stimuli including DNA damage induced by the topoisomerase I inhibitor, camptothecin. We measured DNA fragmentation induced in isolated nuclei by cytosolic extracts using a filter elution assay. Cytosol from camptothecin-treated HL60 cells induced internucleosomal DNA fragmentation in nuclei from untreated cells. This fragmentation was suppressed by serine protease inhibitors. Serine proteases (trypsin, endoproteinase Glu-C, chymotrypsin A, and proteinase K) and papain by themselves induced DNA fragmentation in naive nuclei. This effect was enhanced in the presence of cytosol from untreated cells. Cysteine protease inhibitors (E-64, leupeptin, Ac-YVAD-CHO [ICE inhibitor]) did not affect camptothecin-induced DNA fragmentation. The apopain/Yama inhibitor, Ac-DEVD-CHO, and the proteasome inhibitor, MG-132, were also inactive both in the cell-free system and in whole cells. Interleukin-1 beta converting enzyme (ICE) or human immunodeficiency virus protease failed to induce DNA fragmentation in naive nuclei. Together, these results suggest that DNA damage activates serine protease(s) which in turn activate(s) nuclear endonuclease(s) during apoptosis in HL60 cells.
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PMID:DNA fragmentation induced by protease activation in p53-null human leukemia HL60 cells undergoing apoptosis following treatment with the topoisomerase I inhibitor camptothecin: cell-free system studies. 880 33

We purified by fractionation on 10-40% glycerol gradients, 26S proteasomes from normal human spermatozoa. These proteasomes, which participate in the ATP-dependent degradation of ubiquitinated proteins, share a similar sedimentation coefficient to those purified from other human tissues. Fluorogenic peptide assays reveal they have chymotrypsin, trypsin and peptidyl-glutamyl-like peptide hydrolysing activities; the chymotrypsin activity is ablated by the specific 26S proteasome inhibitor MG132. Confirmation that these large proteases are 26S proteasomes is provided by detection of the 20S proteasome subunits HC2, XAPC7, RN3 and Z and regulatory ATPases MSS1, TBP1, SUG1 and SUG2 by Western analyses with monoclonal antisera. These antigens are found only in the gradient fractions enriched in proteolytic activities. We have also shown that, although mature spermatozoa from mice have considerably reduced amounts of a ubiquitin-conjugating enzyme (E2) and ubiquitin-protein conjugates in comparison with less mature germ cells, they retain relatively high values of 26S proteasome activity. This suggests that proteasomes may have further roles to play in normal sperm physiology.
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PMID:Purification and characterization of 26S proteasomes from human and mouse spermatozoa. 946 50

For an effective CD8+ cytotoxic T cell response to occur during infection, MHC class I molecules must be loaded with antigenic peptides in the endoplasmic reticulum. The cytosolic factor responsible for peptide generation is believed to be the proteasome, with the TAP heterodimer mediating peptide transport into the endoplasmic reticulum. However, the rate-determining step(s) in this intracellular pathway of Ag presentation is currently unresolved. The availability of a specific and irreversible proteasome inhibitor called lactacystin has enabled us to determine the amount of proteasomes required for the peptide loading of MHC class I molecules in four cell types. In the absence of the IFN-gamma-inducible proteasome subunits LMP2 and LMP7, the trypsin-like (but not the chymotrypsin-like) activity of the proteasome is directly related to MHC class I peptide loading. However, IFN-gamma stimulation or assimilation of catalytic LMP2 and LMP7 subunits into proteasomes causes both chymotrypsin- and trypsin-like activities of the proteasome to become limiting for the loading of class I molecules. Our data suggest that upon full IFN-gamma stimulation, peptide supply by the proteasome is the limiting step in the assembly of MHC class I polypeptides. This mechanism may enable the cell to prevent competition between novel Ags and the pool of endogenous proteins for binding to MHC class I molecules.
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PMID:Proteasome activity limits the assembly of MHC class I molecules after IFN-gamma stimulation. 955 Mar 86

Oxidative stress is associated with important pathophysiological events in a variety of diseases. It has been postulated that free radicals and lipid peroxidation products generated during the process may be responsible for these effects because of their ability to damage cellular components such as membranes, proteins, and DNA. In the present study, we provide evidence that oxidative stress causes a transient impairment of intracellular proteolysis via covalent binding of 4-hydroxy-2-nonenal (HNE), a major end product of lipid peroxidation, to proteasomes. A single intraperitoneal treatment with the renal carcinogen, ferric nitrilotriacetate, caused oxidative stress, as monitored by accumulation of lipid peroxidation products and 8-hydroxy-2'-deoxyguanosine, in the kidney of mice. In addition, transient accumulation of HNE-modified proteins in the kidney was also found by competitive enzyme-linked immunosorbent assay and immunohistochemical analyses. This and the observation that the HNE-modified proteins were significantly ubiquitinated suggested a crucial role of proteasomes in the metabolism of HNE-modified proteins. In vitro incubation of the kidney homogenates with HNE indeed resulted in a transient accumulation of HNE-modified proteins, whereas the proteasome inhibitor significantly suppressed the time-dependent elimination of HNE-modified proteins. We found that, among three proteolytic activities (trypsin, chymotrypsin, and peptidylglutamyl peptide hydrolase activities) of proteasomes, both trypsin and peptidylglutamyl peptide hydrolase activities in the kidney were transiently diminished in accordance with the accumulation of HNE-modified proteins during oxidative stress. The loss of proteasome activities was partially ascribed to the direct attachment of HNE to the protein, based on the detection of HNE-proteasome conjugates by an immunoprecipitation technique. These results suggest that HNE may contribute to the enhanced accumulation of oxidatively modified proteins via an impairment of ubiquitin/proteasome-dependent intracellular proteolysis.
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PMID:4-Hydroxy-2-nonenal-mediated impairment of intracellular proteolysis during oxidative stress. Identification of proteasomes as target molecules. 1044 39

The proteasome regulates cellular processes as diverse as cell cycle progression and NF-kappaB activation. In this study, we show that the potent antitumor natural product epoxomicin specifically targets the proteasome. Utilizing biotinylated-epoxomicin as a molecular probe, we demonstrate that epoxomicin covalently binds to the LMP7, X, MECL1, and Z catalytic subunits of the proteasome. Enzymatic analyses with purified bovine erythrocyte proteasome reveal that epoxomicin potently inhibits primarily the chymotrypsin-like activity. The trypsin-like and peptidyl-glutamyl peptide hydrolyzing catalytic activities also are inhibited at 100- and 1,000-fold slower rates, respectively. In contrast to peptide aldehyde proteasome inhibitors, epoxomicin does not inhibit nonproteasomal proteases such trypsin, chymotrypsin, papain, calpain, and cathepsin B at concentrations of up to 50 microM. In addition, epoxomicin is a more potent inhibitor of the chymotrypsin-like activity than lactacystin and the peptide vinyl sulfone NLVS. Epoxomicin also effectively inhibits NF-kappaB activation in vitro and potently blocks in vivo inflammation in the murine ear edema assay. These results thus define epoxomicin as a novel proteasome inhibitor that likely will prove useful in exploring the role of the proteasome in various in vivo and in vitro systems.
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PMID:Epoxomicin, a potent and selective proteasome inhibitor, exhibits in vivo antiinflammatory activity. 1046 20

Lovastatin and simvastatin are HMG-CoA reductase inhibitors widely used as antihyperlipidemic drugs, which also display antiproliferative properties. In the present paper, we provide evidence that both lovastatin and simvastatin are modulators of the purified bovine pituitary 20 S proteasome, since they mildly stimulate the chymotrypsin-like activity and inhibit the peptidylglutamylpeptide hydrolyzing activity without interfering with the trypsin-like activity. However, those effects are only observed when the closed ring forms of the drugs are used, while the opened ring form of lovastatin acts as a mild inhibitor of the chymotrypsin like activity. The closed ring form of lovastatin is much more potent as a cytotoxic agent on the Colon-26 (C-26) colon carcinoma cell line than the opened ring form, which is only mildly cytostatic. Moreover, neither the cytotoxic effects nor the effects on 20 S proteasome activities are prevented by mevalonate, which by itself inhibits the trypsin-like activity of the proteasome. Neither the opened ring nor the closed ring form of lovastatin induces an accumulation of ubiquitin-protein conjugates, which is observed after treatment with lactacystin, a selective proteasome inhibitor. In contrast with the opened ring form of lovastatin, the closed ring form induces the disappearance of detectable p27(kip1) from C-26 cells. Altogether, our results indicate that the closed ring form of lovastatin induces cytotoxic effects independent of its HMG-CoA inhibiting activity, however, those effects are mediated by a complex modulation of proteasome activity rather than by inhibition of the 20 S proteasome.
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PMID:Lovastatin and simvastatin are modulators of the proteasome. 1108 75

Intra-acinar cell nuclear factor-kappaB (NF-kappaB) and trypsinogen activation are early events in secretagogue-induced acute pancreatitis. We have studied the relationship between NF-kappaB and trypsinogen activation in rat pancreas. CCK analogue caerulein induces early (within 15 min) parallel activation of both NF-kappaB and trypsinogen in pancreas in vivo as well as in pancreatic acini in vitro. However, NF-kappaB activation can be induced without trypsinogen activation by lipopolysaccharide in pancreas in vivo and by phorbol ester in pancreatic acini in vitro. Stimulation of acini with caerulein after 6 h of culture results in NF-kappaB but not trypsinogen activation. Protease inhibitors (AEBSF, TLCK, and E64d) inhibit both intracellular trypsin activity and NF-kappaB activation in caerulein stimulated acini. A chymotrypsin inhibitor (TPCK) inhibits NF-kappaB activation but not trypsin activity. The proteasome inhibitor MG-132 prevents caerulein-induced NF-kappaB activation but does not prevent trypsinogen activation. These findings indicate that although caerulein-induced NF-kappaB and trypsinogen activation are temporally closely related, they are independent events in pancreatic acinar cells. NF-kappaB activation per se is not required for the development of early acinar cell injury by supramaximal secretagogue stimulation.
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PMID:Relationship between NF-kappaB and trypsinogen activation in rat pancreas after supramaximal caerulein stimulation. 1116 28

The cytoplasmic trafficking of the prototype strain of minute virus of mice (MVMp) was investigated by analyzing and quantifying the effect of drugs that reduce or abolish specific cellular functions on the accumulation of viral macromolecules. With this strategy, it was found that a low endosomal pH is required for the infection, since bafilomycin A(1) and chloroquine, two pH-interfering drugs, were similarly active against MVMp. Disruption of the endosomal network by brefeldin A interfered with MVMp infection, indicating that viral particles are routed farther than the early endocytic compartment. Pulse experiments with endosome-interfering drugs showed that the bulk of MVMp particles remained in the endosomal compartment for several hours before its release to the cytosol. Drugs that block the activity of the proteasome by different mechanisms, such as MG132, lactacystin, and epoxomicin, all strongly blocked MVMp infection. Pulse experiments with the proteasome inhibitor MG132 indicated that MVMp interacts with cellular proteasomes after endosomal escape. The chymotrypsin-like but not the trypsin-like activity of the proteasome is required for the infection, since the chymotrypsin inhibitors N-tosyl-L-phenylalanine chloromethyl ketone and aclarubicin were both effective in blocking MVMp infection. However, the trypsin inhibitor Nalpha-p-tosyl-L-lysine chloromethyl ketone had no effect. These results suggest that the ubiquitin-proteasome pathway plays an essential role in the MVMp life cycle, probably assisting at the stages of capsid disassembly and/or nuclear translocation.
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PMID:Cytoplasmic trafficking of minute virus of mice: low-pH requirement, routing to late endosomes, and proteasome interaction. 1243 89

We have developed a novel LPS probe using a highly purified and homogenous preparation of [(3)H] Escherichia coli LPS from the deep rough mutant, which contains a covalently linked, photoactivable 4-p-(azidosalicylamido)-butylamine group. This cross-linker was used to identify the LPS-binding proteins in membranes of the murine-macrophage-like cell line RAW 264.7. The alpha-subunit (PSMA1 C2, 29.5 kDa) and the beta-subunit (PSMB4 N3, 24.36 kDa) of the 20S proteasome complex were identified as LPS-binding proteins. This is the first report demonstrating LPS binding to enzymes such as the proteasome subunits. Functionally, LPS enhanced the chymotrypsin-like activity of the proteasome to degrade synthetic peptides in vitro and, conversely, the proteasome inhibitor lactacystin completely blocked the LPS-induced proteasome's chymotrypsin activity as well as macrophage TNF-alpha secretion and the expression of multiple inflammatory mediator genes. Lactacystin also completely blocked the LPS-induced expression of Toll-like receptor 2 mRNA. In addition, lactacystin dysregulated mitogen-activated protein kinase phosphorylation in LPS-stimulated macrophages, but failed to inhibit IL-1 receptor-associated kinase-1 activity. Importantly, lactacystin also prevented LPS-induced shock in mice. These data strongly suggest that the proteasome complex regulates the LPS-induced signal transduction and that it may be an important therapeutic target in Gram-negative sepsis.
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PMID:The proteasome as a lipopolysaccharide-binding protein in macrophages: differential effects of proteasome inhibition on lipopolysaccharide-induced signaling events. 1287 45

Mutations in familial Parkinson's disease (PD) have been associated with the failure of protein degradation through the ubiquitin-proteasome system (UPS). Impairment of proteasome function has also been suggested to play a role in the pathogenesis of sporadic PD. We examined the proteasome activity in PC12 cells treated with 6-hydroxydopamine (6-OHDA), the dopamine synthetic derivate used in models of PD. We found that 6-OHDA treatment increased protein oxidation, as indicated by carbonyl group accumulation, and increased caspase-3 activity. In addition, there was an increase in trypsin-, chymotrypsin-, and postacidic-like proteasome activities in cells treated with 10-100 microM 6-OHDA, whereas higher doses caused a marked decline. 6-OHDA exposure also increased mRNA expression of the 19S regulatory subunit in a dose-dependent manner, whereas the expression of 20S- and 11S-subunit mRNAs did not change. Administration of the antioxidant N-acetylcysteine to 6-OHDA-treated cells prevented the alteration in proteasome functions. Moreover, reduction in cell viability owing to administration of proteasome inhibitor MG132 or lactacystin was partially prevented by the endogenous antioxidant-reduced glutathione. In conclusion, our data indicate that mild oxidative stress elevates proteasome activity in response to increase in protein damage. Severe oxidative insult might cause UPS failure, which leads to protein aggregation and cell death. Moreover, in the case of UPS inhibition or failure, the blockade of physiological reactive oxygen species production during normal aerobic metabolism is enough to ameliorate cell viability. Control of protein clearance by potent, brain-penetrating antioxidants might act to slow down the progression of PD.
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PMID:Oxidative stress, induced by 6-hydroxydopamine, reduces proteasome activities in PC12 cells: implications for the pathogenesis of Parkinson's disease. 1565 61


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