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)

The 20S proteasome purified from animal cells has various latent peptidase activities. Fatty acids such as linoleic, linolenic and oleic acids strongly activate both the chymotrypsin-type and peptidylglutamylpeptide (PGP) hydrolase-type activities, but have been reported to have little activation or inhibition of the trypsin-type activity. We show here that an increase of the fatty acid concentration produces activation of chymotrypsin-type and PGP hydrolase-type in a biphasic fashion: no effect until the threshold concentration and then a sharp activation. In contrast, the trypsin-type activity was markedly inhibited at low concentrations of fatty acid, slightly activated at higher concentrations, and inhibited again at even higher concentrations. The inhibition was removed when the concentration of fatty acid was reduced by dilution after pre-incubation with the fatty acid. As a result, the activation pattern became biphasic, which was identical to that of chymotrypsin-type and PGP hydrolase-type activities. These results suggest that in the chymotrypsin-type and PGP hydrolase-type peptidase fatty acids bind first to a class of sites without direct effect on the peptidase activity, but after saturation of this class it permits more fatty acid to bind to another class of sites involved in the activation. In the trypsin-type peptidase an additional class of fatty acid binding sites is uniquely present, which is involved in the enzyme inhibition. The dilution procedure described above removes the fatty acid molecules bound to the inhibition sites, but not the fatty acid molecules bound to the activation sites; this results in the fatty acid activation profile indistinguishable from that of the chymotrypsin- and PGP hydrolase-type peptidases.
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PMID:Irreversible potent activation and reversible inhibition of trypsin-like activity of 20S proteasome purified from Xenopus oocytes by fatty acid. 961 17

Accumulation of altered proteins in old animals has been ascribed to slower turnover of proteins. Since proteasomes can be regarded as the major proteolytic enzymes responsible for the degradation of the majority of cellular proteins, we examined age-related changes of 20S and 26S proteasomes in the liver of young (8-10-month-old), middle-aged (15-18-month-old) and old (25-28-month-old) Fischer 344 male rats. The two forms of proteasomes were separated by glycerol gradient centrifugation. Fluorogenic peptides were used as substrates to evaluate three types of peptidase activities. The ratio of peptidase activities in the 20S proteasome vs. those in the 26S form did not appear to change with age. Unstimulated chymotrypsin-like activity found only in the 26S form decreased by 30% in the old rats as compared with that in the young ones, while no change in the activity was observed during aging when stimulated by sodium dodecyl sulfate. The trypsin-like activity declined significantly by 17% to an apparently similar extent in both 20S and 26S forms. The peptidylglutamyl peptide hydrolyzing activity exhibited gradual decrease with age, resulting in 60% lower value in the old rats as compared with the young animals. These changes are considered to account for the age-related extension of half-life of proteins. Since the amount of total proteasomes measured by immunoblot did not appear to change with age, posttranslational modifications or subunit replacement is possibly responsible for the decrease in the activities.
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PMID:Age-related changes in the 20S and 26S proteasome activities in the liver of male F344 rats. 966 92

The effects of an activator, cardiolipin, on the three peptidase activities of the 20S proteasome of Xenopus oocytes were examined. The trypsin-like activity was activated when the enzyme was treated with cardiolipin before the addition of the substrate, but there was no appreciable activation when cardiolipin was added concomitantly with the substrate. On the other hand, the chymotrypsin-like peptidase and peptidylglutamylpeptide hydrolase (PGPH) were activated regardless of the sequence of addition. When very low concentrations of the substrate (e.g. 0.1-0.5 microM; about 1/100 of the K(m)) were used, cardiolipin strongly activated trypsin-like peptidase by the simultaneous addition but not after substrate addition. These results suggest that the trypsin-type substrate produces a conformational change in the enzyme in a concentration-dependent manner which makes the activator sites inaccessible to cardiolipin.
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PMID:Activation of the 20S proteasome of Xenopus oocytes by cardiolipin: blockage of the activation of trypsin-like activity by the substrate. 969 14

Ethanol consumption slows down the rate of hepatic protein catabolism. The present study was conducted to determine whether ethanol consumption, given by voluntary (pair) feeding or by intragastric administration, affected the peptidase activities of the proteasome in rat liver. Rats were pair-fed liquid diets containing either ethanol or isocaloric maltose-dextrin. A separate group of animals was intragastrically infused continuously with similar liquid diets containing either ethanol or isocaloric dextrose. Crude liver homogenates and their cytosolic fractions were assayed for their chymotrypsin-like (Cht-L), trypsin-like (T-L), and peptidyl-glutamyl-peptide hydrolase (PGPH) activities, using specific fluorogenic peptides as substrates. Voluntary ethanol feeding did not affect the three peptidase activities of the proteasome. However, intragastric ethanol administration caused a 35% to 40% decline in the Cht-L and the T-L activities, but did not significantly change the PGPH activity. The lower peptidase activities in cytosol samples from intragastrically ethanol-fed rats were not restored to control levels by overnight dialysis, nor by the inclusion of low levels of sodium dodecyl sulfate (SDS) or of 0.5 mmol/L adenosine triphosphate (ATP) in the proteasome assay mixture. Immunoblot analyses using anti-rat liver proteaseome exhibited equal levels of immunoreactive proteasome subunits in livers of control and ethanol-fed rats. Similar results were obtained when blots were probed with antibody made specifically against the proteasome subunit, LMP-7. The results indicate that intragastric, but not voluntary, ethanol consumption differentially affects the separate catalytic activities of the proteasome without affecting its steady-state levels. Such changes may be related to the degree of ethanol-induced oxidative stress.
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PMID:Peptidase activities of the multicatalytic protease in rat liver after voluntary and intragastric ethanol administration. 969 15

A family of ATPases resides within the regulatory particle of the proteasome. These proteins (Rpt1-Rpt6) have been proposed to mediate substrate unfolding, which may be required for translocation of substrates through the channel that leads from the regulatory particle into the proteolytic core particle. To analyze the role of ATP hydrolysis in protein breakdown at the level of the individual ATPase, we have introduced equivalent site-directed mutations into the ATPbinding motif of each RPT gene. Non-conservative substitutions of the active-site lysine were lethal in four of six cases, and conferred a strong growth defect in two cases. Thus, the ATPases are not functionally redundant, despite their multiplicity and sequence similarity. Degradation of a specific substrate can be inhibited by ATP-binding-site substitutions in many of the Rpt proteins, indicating that they co-operate in the degradation of individual substrates. The phenotypic defects of the different rpt mutants were strikingly varied. The most divergent phenotype was that of the rpt1 mutant, which was strongly growth defective despite showing no general defect in protein turnover. In addition, rpt1 was unique among the rpt mutants in displaying a G1 cell-cycle defect. Proteasomes purified from an rpt2 mutant showed a dramatic inhibition of peptidase activity, suggesting a defect in gating of the proteasome channel. In summary, ATP promotes protein breakdown by the proteasome through multiple mechanisms, as reflected by the diverse phenotypes of the rpt mutants.
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PMID:Active site mutants in the six regulatory particle ATPases reveal multiple roles for ATP in the proteasome. 972 28

The proteolytic activity of the eukaryotic 20S proteasome is stimulated by a multisubunit activator, PA700, which forms both 1:1 and 2:1 complexes with the proteasome. Formation of the complexes is enhanced by an additional protein assembly called modulator, which also stimulates the enzymatic activity of the proteasome only in the presence of PA700. Here we show that the binding of PA700 to the proteasome is cooperative, as is the activation of the proteasome's intrinsic peptidase activity. Modulator increases the extent of complex formation and peptidase activation, while preserving the cooperative kinetics. Furthermore, the increase in activity is not linear with the number of PA700 assemblies bound to the proteasome, but rather with the number of proteasome-PA700 complexes, regardless of the PA700:proteasome stoichiometry. Hence the stimulation of peptidase activity is fully (or almost fully) effected by the binding of a single PA700 to the 20S proteasome. The stimulation of peptidase by modulator is explained entirely by the increased number of proteasome-PA700 complexes formed in its presence, rather than by any substantial direct stimulation of catalysis. These observations are consistent with a model in which PA700, either alone or assisted by modulator, promotes conformational changes in the proteasome that activate the catalytic sites and/or facilitate access of peptide substrates to these sites.
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PMID:Formation of proteasome-PA700 complexes directly correlates with activation of peptidase activity. 973 72

An alanyl-alanyl-phenylalanyl-7-amino-4-methylcoumarin-hydrolyzing protease particle copurifying with 26S proteasomes was isolated and identified as tripeptidyl peptidase II (TPPII), a cytosolic subtilisin-like peptidase of unknown function. The particle is larger than the 26S proteasome and has a rod-shaped, dynamic supramolecular structure. TPPII exhibits enhanced activity in proteasome inhibitor-adapted cells and degrades polypeptides by exo- as well as predominantly trypsin-like endoproteolytic cleavage. TPPII may thus participate in extralysosomal polypeptide degradation and may in part account for nonproteasomal epitope generation as postulated for certain major histocompatibility complex class I alleles. In addition, TPPII may be able to substitute for some metabolic functions of the proteasome.
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PMID:A giant protease with potential to substitute for some functions of the proteasome. 997 89

Proteasomes interact with a variety of macromolecular ligands that modulate their ability to degrade peptide and protein substrates. The effector PA28 increases the peptidase activities of proteasomes whereas HSP90 and alpha-crystallin inhibit a peptide-hydrolyzing activity. Four monoclonal antibodies were used as probes to detect conformational changes of proteasome subunits. Conformational changes in alpha- or beta-subunits were found upon binding PA28, HSP90, alpha-crystallin, and the substrate casein but not with the peptide substrate analogs calpain inhibitor 1 (Ac-Leu-Leu-norleucinal), calpain inhibitor 2 (Ac-Leu-Leu-methioninal), or MG 132 (N-Cbz-Leu-Leu-leucinal).
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PMID:Conformational changes in the 20S proteasome upon macromolecular ligand binding analyzed with monoclonal antibodies. 998 42

At the early stages of myogenesis, myoblasts fuse to form multinucleated myotubes. This morphological differentiation is the result of dynamic changes in gene regulation and expression. The ubiquitin proteasome-dependent pathway has been reported to play an important role in many aspects of cellular functions such as regulation of growth and cell cycle progression. In this study, we showed that the amount of mRNA's corresponding to the iota subunit of the 20S proteasome, the level of the S4 subunit of the 19S complex and the 20S and 26S proteasomes peptidase activities increased during myoblast fusion. Cell permeable 20S proteasome inhibitor prevented fusion with concomitant accumulation of ubiquitin-conjugated protein. On the other hand, inhibition of ubiquitin ligase E3 enzymes prevented the formation of ubiquitin conjugate and decreased the fusion process. These results strongly support the involvement of the ubiquitin-proteasome proteolytic pathway in the events leading to myoblast fusion.
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PMID:Degradation of an ubiquitin-conjugated protein is associated with myoblast differentiation in primary cell culture. 1020 75

The mechanism of the activation of intracellular proteasomes at fertilization was measured in living sand dollar eggs using the membrane-impermeant fluorogenic substrate, succinyl-Phe-Leu-Arg-coumarylamido-4-methanesulfonic acid. When the substrate was microinjected into unfertilized eggs, the initial velocity of hydrolysis of the substrate (V0) was low. V0 measured 5 to 10 min after fertilization was five to nine times the prefertilization level and remained high throughout the first cell cycle. Hydrolysis of the substrate was inhibited by clasto-lactacystin beta-lactone, a specific inhibitor of the proteasome. There has been in vitro evidence that calcium may be involved in regulation of proteasome activity to either inhibit the increase in peptidase activity associated with PA 28 binding to the 20S proteasome or stimulate activity of the PA 700-proteasome complex. Since both intracellular free Ca2+ concentration ([Ca2+]i) and intracellular pH (pHi) increase after fertilization, hydrolysis of the proteasome substrate was measured under conditions in which [Ca2+]i and pHi were varied independently during activation. When the pHi of unfertilized eggs was elevated by exposure to 15 mM ammonium chloride in pH 9 seawater, V0 increased to a level comparable to that measured after fertilization. In contrast, [Ca2+]i elevation without pHi change, induced by calcium ionophore in sodium-free seawater, had no effect on V0 in the unfertilized egg. Moreover, when unfertilized eggs were microinjected with buffers modulating pHi, V0 increased in a pH-dependent manner. These results indicate that the pHi rise at fertilization is the necessary prerequisite for activation of the proteasome, an essential component in the regulation of the cell cycle.
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PMID:Activation of the proteasomes of sand dollar eggs at fertilization depends on the intracellular pH rise. 1020 42


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