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 proteasome is a multisubunit protease responsible for degrading proteins conjugated to ubiquitin. The 670-kDa core particle of the proteasome contains the proteolytic active sites, which face an interior chamber within the particle and are thus protected from the cytoplasm. The entry of substrates into this chamber is thought to be governed by the regulatory particle of the proteasome, which covers the presumed channels leading into the interior of the core particle. We have resolved native yeast proteasomes into two electrophoretic variants and have shown that these represent core particles capped with one or two regulatory particles. To determine the subunit composition of the regulatory particle, yeast proteasomes were purified and analyzed by gradient sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Resolution of the individual polypeptides revealed 17 distinct proteins, whose identities were determined by amino acid sequence analysis. Six of the subunits have sequence features of ATPases (Rpt1 to Rpt6). Affinity chromatography was used to purify regulatory particles from various strains, each of which expressed one of the ATPases tagged with hexahistidine. In all cases, multiple untagged ATPases copurified, indicating that the ATPases assembled together into a heteromeric complex. Of the remaining 11 subunits that we have identified (Rpn1 to Rpn3 and Rpn5 to Rpn12), 8 are encoded by previously described genes and 3 are encoded by genes not previously characterized for yeasts. One of the previously unidentified subunits exhibits limited sequence similarity with deubiquitinating enzymes. Overall, regulatory particles from yeasts and mammals are remarkably similar, suggesting that the specific mechanistic features of the proteasome have been closely conserved over the course of evolution.
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PMID:The regulatory particle of the Saccharomyces cerevisiae proteasome. 958 56

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

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

We previously reported that ethanol elicits an increased protein oxidation in the liver of rats receiving chronic ethanol by continuous intragastric infusion (Tsukamoto-French method). This accumulation of oxidized proteins could result from a decrease in the cytosolic proteolysis, related specifically to alkaline protease and its major components, the proteasomes. Because several studies suggest that intracellular proteolysis depends on the severity of oxidative stress, we investigated the cytosolic proteolytic activity under two chronic ethanol treatment paradigms associated with varying degrees of oxidative stress. For 4 weeks, male rats received chronic ethanol by continuous intragastric infusion or by oral administration (10% ethanol ad libitum as sole drinking fluid). A significant decrease was evident for alkaline protease activity as well as for sodium dodecyl sulfate (SDS)-activated latent 20S proteasome (chymotrypsine-like [ChT-L] and peptidylglutamyl peptide hydrolase [PGPH] activities) in the liver of rats receiving ethanol by continuous intragastric infusion. Free radical production and related processes appeared to be contributing events in proteolysis inhibition, because phenethyl isothiocyanate (PIC), an inhibitor of cytochrome P4502E1 (CYP2E1), reduced the inhibition of the ethanol-related ChT-L activity. Moreover, the lipid peroxidation level was inversely correlated with ChT-L activity. In contrast, no such changes were observed in ChT-L and PGPH activities or in cellular free radical targets following the oral ad libitum consumption of 10% ethanol. It appears, thus, that only the alcohol treatment paradigm associated with an overt oxidative stress produced a significant inhibition of the proteasome activity. The mechanisms of proteasome inhibition could involve the formation of an endogenous inhibitor such as protein aggregates or aldehyde-derivative peptides. Whatever the mechanism, the inhibition of cytosolic proteolysis and the subsequent accumulation of damaged proteins may be involved in the oxidatively challenged alcoholic livers and play a pathogenic role in experimental alcoholic liver disease.
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PMID:Effects of chronic ethanol administration on rat liver proteasome activities: relationship with oxidative stress. 986 43

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

Pig membrane cofactor protein (MCP; CD46) is a 50 000-60 000 MW glycoprotein that is expressed on a wide variety of cells, including erythrocytes. Pig MCP has cofactor activity for factor I-mediated cleavage of C3b and is an efficient regulator of the classical and alternative pathway of human and pig complement. A panel of 10 monoclonal antibodies (mAbs) was collected from two different laboratories; all of these mAbs were raised against pig leucocytes and all recognized the same complex banding pattern on sodium dodecyl sulphate-polyacrylamide gel electrophoresis (SDS-PAGE) of erythrocyte membranes. All were shown to be reactive with pig MCP and were divided into four groups of mutually competitive antibodies based on competition studies for membrane-bound MCP and for soluble MCP, the latter by surface plasmon resonance (SPR) analysis. The antigenic properties of membrane-bound and soluble MCP were similar, although some interesting differences were revealed. None of the 10 mAbs were cross-reactive with human MCP and only one showed cross-reactivity with leucocytes from a panel of large mammals - a weak cross-reactivity with a subset of dog leucocytes. All antibodies in one of the epitope groups and some in a second epitope group were able to block the functional activity of pig MCP, as measured by inhibition of MCP-catalysed C3 degradation by factor I.
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PMID:Epitope mapping of 10 monoclonal antibodies against the pig analogue of human membrane cofactor protein (MCP). 1023 56

The ability of ethanol to inhibit regenerative processes in the liver is thought to play a key role in the development of alcoholic liver disease. To understand the underlying mechanisms, we investigated the effects of ethanol on the Janus kinasesignal transducer and activator transcription factor (JAK-STAT) signaling pathways in hepatocytes. Treatment of freshly isolated adult rat hepatocytes with 10-100 mM ethanol rapidly (< 3 min) inhibits interleukin-6 (IL-6)-induced STAT3 activation, tyrosine and serine phosphorylation and IL-6-induced CCAAT enhancer binding protein (C/EBP) alpha and beta mRNA expression. Western analyses, in vitro kinase assays and in vivo cell labelling assays indicate that this inhibitory effect is not due to blocking the upstream-located JAK1, JAK2 or Tyk2 activation. On the contrary, acute ethanol exposure significantly potentiates IL-6-induced JAK1 autophosphorylation in vitro and in vivo. Pretreatment with sodium vanadate, a non-selective tyrosine phosphatase inhibitor, or with MG132 and lactacystin, proteasome inhibitors, does not abolish the ethanol inhibition of IL-6-induced STAT3 activation, suggesting that activation of protein tyrosine phosphatases or the ubiquitin-proteasome pathway is not involved. In view of the critical role of IL-6 signaling in liver regeneration, these findings suggest that the ability of biologically relevant concentrations of ethanol to markedly inhibit IL-6-induced STAT3 phosphorylation is one of the cellular mechanisms involved in the pathogenesis and progression of alcoholic liver diseases.
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PMID:Ethanol rapidly inhibits IL-6-activated STAT3 and C/EBP mRNA expression in freshly isolated rat hepatocytes. 1048 86

Two new forms of proteasomes, designated as the endoplasmic reticulum (ER) membrane-associated proteasome (ERa proteasome) and ER membrane-bound proteasome (ERb proteasome), were purified to homogeneity from 0.0125 and 2.5% sodium cholate extracts, respectively, of a rat liver microsomal fraction. SDS-PAGE analysis revealed that the purified ERa and ERb proteasomes were composed of multiple subunits similar to the cytosolic 20S proteasome. However, electrophoretic, structural and immunochemical differences between the ERa, ERb and cytosolic 20S proteasomes were observed on native PAGE, two-dimensional (2D) PAGE, and immunoblot analyses. Purification of ERb from a 2.5% sodium cholate extract of the trypsin-treated microsomal fraction yielded a trypsin-modified form of ERb (tERb), which lacked the C2 subunit at least. On the other hand, no ERa proteasome was obtained from the 0.0125% sodium cholate extract of the trypsin-treated microsomes, suggesting that ERa and ERb are ER membrane-associated and -bound proteasomes, respectively. The ERa, ERb, and cytosolic 20S proteasomes exhibited similar specificities as to peptide hydrolyzing activity, although differences in their activities were noted in the presence of SDS and phospholipid. With respect to the proteolysis of protein substrates, only the ERb proteasome cleaved beta-casein, and it also degraded reduced and carboxymethylated lysozyme considerably faster than the cytosolic 20S and ERa proteasomes. Collectively our results suggest that the ERa and ERb proteasomes may play roles in intracellular proteolysis distinct from that of the cytosolic 20S proteasome.
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PMID:Isolation and characterization of two 20S proteasomes from the endoplasmic reticulum of rat liver microsomes. 1050 81

The mechanisms of cadmium (Cd)-dependent nephrotoxicity were studied in a rat proximal tubule (PT) cell line. CdCl(2) (5 microM) increased the production of reactive oxygen species (ROS), as determined by oxidation of dihydrorhodamine 123 to fluorescent rhodamine 123. The levels of ubiquitin-conjugated cellular proteins were increased by Cd in a time-dependent fashion (maximum at 24-48 h). This was prevented by coincubation with the thiol antioxidant N-acetylcysteine (NAC, 15 mM). Cd also increased apoptosis (controls: 2.4+/-1.6%; Cd: 8.1+/-1.9%), but not necrosis (controls: 0.5 +/- 0.3%; Cd: 1.4+/- 2.5%). Exposure of PT cells with Cd decreased protein levels of the catalytic subunit (alpha1) of Na+/K(+)-ATPase, a long-lived membrane protein (t(1/2)>48 h) that drives reabsorption of ions and nutrients through Na(+)-dependent transporters in PT. Incubation of PT cells for 48 h with Cd decreased Na+/K(+)-ATPase alpha1-subunit, as determined by immunoblotting, by approximately 50%, and NAC largely prevented this effect. Inhibitors of the proteasome such as MG-132 (20 microM) or lactacystin (10 microM), as well as lysosomotropic weak bases such as chloroquine (0.2 mM) or NH(4)Cl (30 mM), significantly reduced the decrease of Na(+)/K(+)-ATPase alpha1-subunit induced by Cd, and in combination abolished the effect of Cd on Na+/K(+)-ATPase. Immunofluorescence labeling of Na+/K(+)-ATPase showed a reduced expression of the protein in the plasma membrane of Cd-exposed cells. After addition of lactacystin and chloroquine to Cd-exposed PT cells, immunoreactive material accumulated into intracellular vesicles. The data indicate that micromolar concentrations of Cd can increase ROS production and exert a toxic effect on PT cells. Oxidative damage increases the degradation of Na+/K(+)-ATPase through both the proteasomal and endo-/lysosomal proteolytic pathways. Degradation of oxidatively damaged Na+/K(+)-ATPase may contribute to the 'Fanconi syndrome'-like Na(+)-dependent transport defects associated with Cd-nephrotoxicity.
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PMID:Cadmium-mediated oxidative stress in kidney proximal tubule cells induces degradation of Na+/K(+)-ATPase through proteasomal and endo-/lysosomal proteolytic pathways. 1050 78

Cellular iron storage and uptake are coordinately regulated post-transcriptionally by cytoplasmic factors, iron-regulatory proteins 1 and 2 (IRP-1 and IRP-2). When iron in the intracellular transit pool is scarce, IRPs bind to iron-responsive elements (IREs) in the 5'-untranslated region of the ferritin mRNA and 3'-untranslated region of the transferrin receptor (TfR) mRNA. Such binding inhibits translation of ferritin mRNA and stabilizes the mRNA for TfR, whereas the opposite scenario develops when iron in the transit pool is plentiful. However, we (Richardson, D. R., Neumannova, V., Nagy, E., and Ponka, P. (1995) Blood 86, 3211-3219) and others reported that the binding of IRPs to IREs can also be modulated by nitric oxide (NO). In this study, we showed that a short exposure of RAW 264.7 cells (a murine macrophage cell line) to the NO(+) donor, sodium nitroprusside (SNP), caused a significant decrease in IRP-2 binding to the IREs followed by IRP-2 degradation and that these changes occurred without affecting IRP-1 binding. The SNP-mediated degradation of IRP-2 in RAW 264.7 cells could be prevented by MG-132 or lactacystin, known inhibitors of proteasome-dependent protein degradation. A SNP-mediated decrease in IRP-2 binding and levels was associated with a dramatic decrease in TfR mRNA levels and an increase in ferritin synthesis. Importantly, the proteasome inhibitor MG-132 prevented the SNP-mediated decrease in TfR mRNA levels. These observations suggest that IRP-2 can play an important role in controlling transferrin receptor expression.
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PMID:Control of transferrin receptor expression via nitric oxide-mediated modulation of iron-regulatory protein 2. 1055 72


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