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)

An inhibitory protein for the 20S proteasome (also known as macropain, the multicatalytic proteinase complex and 20S proteinase) has been purified from bovine red blood cells. The inhibitor has an apparent molecular weight of 31,000 on SDS-PAGE and appears to form multimers under nondenaturing conditions. This protein inhibited all three of the putatively distinct catalytic activities of proteasome A (the active form of the proteinase) characterized by the hydrolysis of synthetic peptides such as Z-VLR-MNA, Z-GGL-AMC or Suc-LLVY-AMC and Z-LLE-beta NA. The inhibitor also prevented the hydrolysis of large protein substrates such as casein, lysozyme and bovine serum albumin. Proteasome L (the latent form of the proteinase) does not degrade these large protein substrates, but does hydrolyze the three synthetic peptides at rates similar to those by proteasome A. The inhibitor inhibited only two of these peptidase activities of proteasome L (hydrolysis of Z-GGL-AMC and of Z-LLE-beta NA or Suc-LLVY-AMC); it had no effect on the hydrolysis of Z-VLR-MNA. The inhibitor was specific for inhibition of the proteasome and had no effect on the activity of any other proteinase tested including trypsin, chymotrypsin, papain, subtilisin and both isoforms of calpain. Kinetic analysis indicates that the inhibitor interacted with the proteasome by a mechanism involving tight-binding. Because the proteasome appears to be a key component of the ATP/ubiquitin-dependent pathway of intracellular protein degradation, the inhibitor may represent an important regulatory protein of this pathway.
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PMID:Purification and characterization of a protein inhibitor of the 20S proteasome (macropain). 131 59

Proteasome, a high molecular weight multicatalytic protease, was purified from the cytosolic fraction of human platelets for the first time. The biochemical properties of the enzyme including substrate specificity, optimal pH and effects of various inhibitors were almost identical with those of other cells. During the purification with a Heparin-Sepharose chromatography, a novel endogenous activator of the protease was identified and was partially purified. The activator enhanced both chymotrypsin or trypsin like activities of the proteasome in a dose related manner and was inactivated by heating at 56 degrees C for 30 min. This newly identified activator may serve as an important regulator or cofactor of intracellular activities of the proteasome.
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PMID:Proteasome and its novel endogeneous activator in human platelets. 206 66

Aged Lou female rats (33 months) submitted to a self-selection regimen showed a decrease in protein intake (down to 11% of the total intake), whereas mature rats (18 months) selected a high percentage of protein (20% of the total intake) similar to the protein content of the standard diet. To find out if this decrease in protein intake would prevent an observed age-related decrease in proteasome activity, four peptidase activities and oxidized protein degradation were tested with proteasome purified from the liver of 18- and 33-month-old rats. The peptidylglutamyl-peptide hydrolase activity, which is decreased with age for rats fed the standard diet, was restored in the self-selecting old rats to the level observed for the mature rats. Degradation of oxidized glutamine synthetase, which is also decreased with age for rats fed the standard diet, was partly restored. Proteasome from self-selecting old rats showed a slight increase in trypsin-like and chymotrypsin-like activities as compared to proteasome from old rats fed the standard diet. Two-dimensional gel electrophoresis followed by quantitative analysis of the pattern of proteasome subunits revealed an increase in the intensity of two protein spots for proteasome from old rats fed the standard diet as compared with proteasome from either mature rats or self-selecting old rats. These findings may have important implications in aging for proteasome-mediated proteolysis and subsequent accumulation of oxidatively damaged protein.
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PMID:Dietary self-selection can compensate an age-related decrease of rat liver 20 S proteasome activity observed with standard diet. 959 40

The hepatitis B virus X protein (HBX) is essential for the establishment of HBV infection in vivo and exerts a pleiotropic effect on diverse cellular functions. The yeast two-hybrid system had indicated that HBX could interact with two subunits of the 26S proteasome. Here we demonstrate an association in vivo of HBX with the 26S proteasome complex by coimmunoprecipitation and colocalization upon sucrose gradient centrifugation. Expression of HBX in HepG2 cells caused a modest decrease in the proteasome's chymotrypsin- and trypsin-like activities and in hydrolysis of ubiquitinated lysozyme, suggesting that HBX functions as an inhibitor of proteasome. In these cells, HBX is degraded with a half-life of 30 min. Proteasome inhibitors retarded this rapid degradation and caused a marked increase in the level of HBX and an accumulation of HBX in polyubiquitinated form. Thus, the low intracellular level of HBX is due to rapid proteolysis by the ubiquitin-proteasome pathway. Surprisingly, the proteasome inhibitors blocked the transactivation by HBX, and this effect was not a result of a squelching phenomenon due to HBX accumulation. Therefore, proteasome function is possibly required for the transactivation function of HBX. The inhibition of protein breakdown by proteasomes may account for the multiple actions of HBX and may be an important feature of HBV infection, possibly in helping stabilize viral gene products and suppressing antigen presentation.
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PMID:Hepatitis B virus X protein is both a substrate and a potential inhibitor of the proteasome complex. 1043 10

Recent evidence supports a role for heat-shock protein 70 (hsp70) and the 26 S proteasome in regulating apoptosis, although the precise nature of their involvement is not known. In the present study, control and Bcl-x(L)-overexpressing, interleukin-3-dependent FL5.12 cell lines were treated with the proteasome inhibitor N-benzoyloxycarbonyl (Z)-Leu-Leu-leucinal (MG132). Basal proteasome activity appeared to be approximately 30% lower in bcl-x(L) cells compared with control cells using a substrate for the chymotrypsin-like activity. However, no difference in proteasome activity was detected using substrates for the trypsin-like or peptidylglutamyl peptide-hydrolysing activities. In addition, protein levels of the 20 S proteasome beta-subunit, as determined by Western blot analyses, were similar in control and bcl-x(L) cells, leading to the conclusion that proteasome activities were the same in these two cell lines. At 24 h after treatment with 500 nM MG132, apoptosis in bcl-x(L) cells (22%) was less than that observed in control cells (34%). Concomitantly, caspase activity in control cells, as assessed by N-acetyl-l-aspartyl-l-glutamyl-l-valyl-l-aspartyl-7-amino-4-methylcou marin (Ac-DEVD-AMC), was twice that observed in bcl-x(L) cells. By 48 h after MG132 treatment, apoptosis and caspase activity in bcl-x(L) cells were similar to those observed in control cells at 24 h. Proteasome inhibition stimulated increases in hsp70 protein levels in control and bcl-x(L) cells by 12 h, although the maximal increases found in bcl-x(L) cells were less. Blocking this induction with hsp70 antisense oligonucleotides potentiated apoptosis after treatment with MG132. Inhibiting caspase activity with a broad-spectrum caspase inhibitor, t-butoxycarbonyl-Asp(OMe)-fluoromethyl ketone, prevented MG132-induced apoptosis. The more specific caspase-3 inhibitor, Ac-DEVD-aldehyde, afforded less protection, although both inhibitors completely inhibited Ac-DEVD-AMC cleavage. These data indicate that both hsp70 and Bcl-x(L) provide some protection against proteasome inhibitor-induced apoptosis.
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PMID:Heat-shock protein 70 antisense oligomers enhance proteasome inhibitor-induced apoptosis. 1056 31

The accumulation of alpha-synuclein, ubiquitin and other proteins in Lewy bodies in degenerating dopaminergic neurones in substantia nigra in idiopathic Parkinson's disease (PD) suggest that inhibition of normal/abnormal protein degradation may contribute to neuronal death. We now show for the first time that the chymotrypsin- (39%), trypsin- (42%) and postacidic-like (33%) hydrolysing activities of 20/26S proteasome are impaired in substantia nigra in PD. Proteasome inhibition does not appear to result from drug treatment since high concentrations of L-3,4-dihydroxyphenylalanine had no effect on enzymatic activity in vitro. These observations provide the first direct evidence that inhibition of the ubiquitin-proteasome pathway leading to altered protein handling and Lewy body formation may be responsible for degeneration of the nigrostriatal pathway in idiopathic PD.
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PMID:Proteasomal function is impaired in substantia nigra in Parkinson's disease. 1113 60

The proteasome inhibitors lactacystin, clastro lactacystin beta-lactone, or tri-leucine vinyl sulfone (NLVS), in the presence of [(35)S]cysteine/methionine, caused increased incorporation of (35)S into cellular proteins, even when protein synthesis was inhibited by cycloheximide. This effect was blocked by incubation with the glutathione synthesis inhibitor buthionine sulfoximine. Proteasome inhibitors also enhanced total glutathione levels, increased reduced/oxidized glutathione ratio (GSH/GSSG) and upregulated gamma-glutamylcysteine synthetase (rate-limiting in glutathione synthesis). Micromolar concentrations of GSH, GSSG, or cysteine stimulated the chymotrypsin-like activity of purified 20S proteasome, but millimolar GSH or GSSG was inhibitory. Interestingly, GSH did not affect 20S proteasome's trypsin-like activity. Enhanced proteasome glutathiolation was verified when purified preparations of the 20S core enzyme complex were incubated with [(35)S]GSH after pre-incubation with any of the inhibitors. NLVS, lactacystin or clastro lactacystin beta-lactone may promote structural modification of the 20S core proteasome, with increased exposure of cysteine residues, which are prone to S-thiolation. Three main conclusions can be drawn from the present work. First, proteasome inhibitors alter cellular glutathione metabolism. Second, proteasome glutathiolation is enhanced by inhibitors but still occurs in their absence, at physiological GSH and GSSG levels. Third, proteasome glutathiolation seems to be a previously unknown mechanism of proteasome regulation in vivo.
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PMID:Glutathiolation of the proteasome is enhanced by proteolytic inhibitors. 1133 15

Proteasomes, the proteolytic machinery of the ubiquitin/ATP-dependent pathway, have a relevant role in many processes crucial for cell physiology and cell cycle progression. Proteasome inhibitors are used to block cell cycle progression and to induce apoptosis in certain cell lines. Here we examine whether proteasomal function is affected by the anti-tumour drug vinblastine, whose cytostatic action relies mainly on the disruption of mitotic spindle dynamics. The effects of vinblastine on the peptidase activities of human 20 S and 26 S proteasomes and on the proteolytic activity of 26 S proteasome were assessed in the presence of specific fluorogenic peptides and (125)I-lysozyme-ubiquitin conjugates respectively. The assays of ubiquitin-protein conjugates and of inhibitory kappa B alpha (I kappa B alpha), which are characteristic intracellular proteasome substrates, by Western blotting on lysates from HL60 cells incubated with or without vinblastine, illustrated the effects of vinblastine on proteasomes in vivo. We also evaluated the effects of vinblastine on the signal-induced degradation of I kappa B alpha. Vinblastine at 3--110 microM reversibly inhibited the chymotrypsin-like activity of the 20 S proteasome and the trypsin-like and peptidyl-glutamyl-peptide hydrolysing activities of both proteasomes, but only at 110 microM vinblastine was the chymotrypsin-like activity of the 26 S proteasome inhibited; furthermore, at 25--200 microM the drug inhibited the degradation of ubiquitinated lysozyme. In HL60 cells exposed for 6 h to 0.5--10 microM vinblastine, the drug-dose-related accumulation of polyubiquitinated proteins, as well as that of a high-molecular-mass form of I kappa B alpha, occurred. Moreover, vinblastine impaired the signal-induced degradation of I kappa B alpha. Cell viability throughout the test was approx. 95%. Proteasomes can be considered to be a new and additional vinblastine target.
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PMID:Proteasomes are a target of the anti-tumour drug vinblastine. 1138 92

Chemotherapy has cachectic effects, but it is unknown whether cytostatic agents alter skeletal muscle proteolysis. We hypothesized that chemotherapy-induced alterations in protein synthesis should result in the increased incidence of abnormal proteins, which in turn should stimulate ubiquitin-proteasome-dependent proteolysis. The effects of the nitrosourea cystemustine were investigated in skeletal muscles from both healthy and colon 26 adenocarcinoma-bearing mice, an appropriate model for testing the impact of cytostatic agents. Muscle wasting was seen in both groups of mice 4 days after a single cystemustine injection, and the drug further increased the loss of muscle proteins already apparent in tumor-bearing animals. Cystemustine cured the tumor-bearing mice with 100% efficacy. Surprisingly, within 11 days of treatment, rates of muscle proteolysis progressively decreased below basal levels observed in healthy control mice and contributed to the cessation of muscle wasting. Proteasome-dependent proteolysis was inhibited by mechanisms that include reduced mRNA levels for 20S and 26S proteasome subunits, decreased protein levels of 20S proteasome subunits and the S14 non-ATPase subunit of the 26S proteasome, and impaired chymotrypsin- and trypsin-like activities of the enzyme. A combination of cisplatin and ifosfamide, two drugs that are widely used in the treatment of cancer patients, also depressed the expression of proteasomal subunits in muscles from rats bearing the MatB adenocarcinoma below basal levels. Thus, a down-regulation of ubiquitin-proteasome-dependent proteolysis is observed with various cytostatic agents and contributes to reverse the chemotherapy-induced muscle wasting.
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PMID:Chemotherapy inhibits skeletal muscle ubiquitin-proteasome-dependent proteolysis. 1201 53

Injury to motor neurons associated with mutant Cu,Zn-superoxide dismutase (SOD1)-related familial amyotrophic lateral sclerosis (FALS) results from a toxic gain-of-function of the enzyme. The mechanisms by which alterations to SOD1 elicit neuronal death remain uncertain despite intensive research effort. Analysis of the cellular proteins that are differentially expressed in the presence of mutant SOD1 represents a novel approach to investigate further this toxic gain-of-function. By using the motor neuron-like cell line NSC34 stably transfected with wild-type, G93A, or G37R mutant human SOD1, we investigated the effects of mutant human SOD1 on protein expression using proteomic approaches. Seven up-regulated proteins were identified as argininosuccinate synthase, argininosuccinate lyase, neuronal nitric-oxide synthase, RNA-binding motif protein 3, peroxiredoxin I, proteasome subunit beta 5 (X), and glutathione S-transferase (GST) Alpha 2. Seven down-regulated proteins were identified as GST Mu 1, GST Mu 2, GST Mu 5, a hypothetical GST Mu, GST Pi B, leukotriene B(4) 12-hydroxydehydrogenase, and proteasome subunit beta5i (LMP7). GST assays demonstrated a significant reduction in the total GST activity of cells expressing mutant human SOD1. Proteasome assays demonstrated significant reductions in chymotrypsin-like, trypsin-like, and post-glutamylhydrolase proteasome activities. Laser capture microdissection of spinal cord motor neurons from human FALS cases, in conjunction with reverse transcriptase-PCR, demonstrated decreased levels of mRNA encoding GST Mu 1, leukotriene B(4) 12-hydroxydehydrogenase, and LMP7. These combined approaches provide further evidence for involvement of alterations in antioxidant defenses, proteasome function, and nitric oxide metabolism in the pathophysiology of FALS.
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PMID:Analysis of the cytosolic proteome in a cell culture model of familial amyotrophic lateral sclerosis reveals alterations to the proteasome, antioxidant defenses, and nitric oxide synthetic pathways. 1247 80


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