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 multicatalytic proteinase complex is a high molecular weight nonlysosomal proteinase. Kinetic studies of the proteolytic activities of the complex have shown that there are at least three distinct types of catalytic centre, each of which has a different specificity. All of the activities can be inhibited by the serine protease inhibitor 3,4-dichloroisocoumarin. Viewed under the electron microscope, the multicatalytic proteinase purified from rat liver appears to have a hollow cylindrical structure. It is composed of many different types of subunit and on two-dimensional polyacrylamide gels gives rise to a complex pattern of about 20 spots with pI values ranging between 5 and 8.5 and molecular masses between 22 and 34 kDa. Immunoblot analysis has shown that many of the major polypeptides are antigenically distinct. However, there are some relationships between the proteinase polypeptides. For example, although N-terminal sequences of five of the polypeptides are unique, they show considerable sequence similarity suggesting that these proteins are encoded by members of the same gene family. Also, there is some cross-reactivity between certain polypeptides when blots are probed with affinity purified, subunit-specific antisera. In addition to the variety of polypeptide components of the proteinase, a small RNA species (80 nucleotides) can be found associated with the complex even after purification by chromatographic procedures.
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PMID:Components of the multicatalytic proteinase complex. 172 5

The 700-kDa multicatalytic proteinase complex from bovine pituitaries separates in polyacrylamide gel electrophoresis under dissociating and reducing conditions into 11 components with molecular masses ranging from 21 to 32 kDa. No higher molecular mass components were detected. A rabbit polyclonal antibody raised against the complex recognizes five immunoreactive components. As reported previously, the complex exhibits three distinct proteolytic activities designated as chymotrypsin-like, trypsin-like, and peptidylglutamyl-peptide hydrolyzing activities. All three activities are rather rapidly inactivated by 3,4-dichloroisocoumarin, a general serine protease inhibitor, however, the pseudo-first-order rate constants of inactivation of the three components differ within a wide range, with the chymotrypsin-like activity being most sensitive to inhibition. The peptidylglutamyl-peptide hydrolyzing activity is greatly activated by low concentrations of sodium dodecyl sulfate and fatty acids and seems to constitute the main component responsible for degradation of protein substrates. In addition to cleaving bonds on the carboxyl side of glutamyl residues, this activity also cleaves, albeit at a slower rate, bonds on the carboxyl side of hydrophobic residues; however, the secondary specificity of this component is clearly different from the chymotrypsin-like activity. Heparin selectively activates the chymotrypsin-like activity. The complex cleaves rapidly both native and dephosphorylated beta-casein in a reaction greatly accelerated by low concentrations of sodium dodecyl sulfate. The nature of proteolytic products, and also the rate of formation of acid-soluble, ninhydrin-reactive products, is different for the phosphorylated and dephosphorylated form of beta-casein, indicating that the degree of phosphorylation influences the rate and pattern of proteolysis.(ABSTRACT TRUNCATED AT 250 WORDS)
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PMID:Pituitary multicatalytic proteinase complex. Specificity of components and aspects of proteolytic activity. 253 72

The catalytic mechanism of the 20S proteasome from the archaebacterium Thermoplasma acidophilum has been analyzed by site-directed mutagenesis of the beta subunit and by inhibitor studies. Deletion of the amino-terminal threonine or its mutation to alanine led to inactivation of the enzyme. Mutation of the residue to serine led to a fully active enzyme, which was over ten times more sensitive to the serine protease inhibitor 3,4-dichloroisocoumarin. In combination with the crystal structure of a proteasome-inhibitor complex, the data show that the nucleophilic attack is mediated by the amino-terminal threonine of processed beta subunits. The conservation pattern of this residue in eukaryotic sequences suggests that at least three of the seven eukaryotic beta-type subunit branches should be proteolytically inactive.
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PMID:Proteasome from Thermoplasma acidophilum: a threonine protease. 772 96

The inhibition of progesterone-induced oocyte maturation by diisopropylfluorophosphate (DFP), a typical serine protease inhibitor, was investigated in oocytes of the Japanese toad Bufo japonicus for the first time. Oocytes to which DFP was externally applied did not undergo germinal vesicle breakdown (GVBD), which is an early signal of oocyte maturation, in response to progesterone. The more inhibitory period was found to be 0-0.5 GVBD50 on a relative time scale [when the time at which 50% of the oocytes had completed GVBD (GVBD50) was set at 1.0], namely, before the beginning of GVBD. DFP-sensitive proteases, which seem to be multifunctional nonlysosomal protease complexes (proteasomes), may already be present in the cytosol of premature oocytes. Peptide hydrolyzing activity, as reflected by proteasome activity, was found to be regulated before and after GVBD. In addition, immunoblotting regarding the native electrophoretic protein profile of the proteasomes throughout the maturational process demonstrated that they undergo alterations in mobility dependent upon the maturational process. These findings raise the possibility that the activities of some endogenous DFP-sensitive proteasomes play distinct, essential roles in oocyte maturation triggered by progesterone in Bufo.
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PMID:DFP-sensitive multicatalytic protease complexes (proteasomes) involved in the control of oocyte maturation in the toad, Bufo japonicus. 791 82

It is well established that the functional properties of proteins can be compromised by oxidative damage and, in vivo, proteins modified by oxidants are rapidly degraded. It was hypothesized that oxidants may also affect the ability of proteases to hydrolyze peptides and proteins. We therefore examined the effect of oxidants on the endopeptidase activities of the 650 kDa 20S proteasome or multicatalytic endopeptidase (MCP), which is thought to play a central role in nonlysosomal protein breakdown. Treatment of the MCP with the oxidant system, FeSO4-EDTA-ascorbate, stimulated the peptidase activities of the MCP while H2O2 treatment showed little or no stimulation. However, treatment of the MCP with FeSO4-EDTA-ascorbate or H2O2 stimulated proteinase activity by 480% and 730%, respectively. An endogenous activator of the MCP, PA28, stimulated the acidic, basic, and hydrophobic peptidase activities of the MCP, but had no effect on proteolytic activity. Treatment of PA28 with oxidants in the presence of MCP or alone did not greatly affect PA28's ability to activate the peptidase activities of the MCP. Using nondenaturing polyacrylamide gel electrophoresis, structural alterations in the enzyme which may be responsible for the activation of peptidase and protease activities following exposure to oxidants were investigated. Treatment of the MCP with reagents that activate proteolysis, including H2O2, as well as the serine protease inhibitor 3,4-dichloroisocoumarin and the cysteine protease inhibitor p-(chloromercuri) benzenesulfonic acid, all caused dissociation of the 650 kDa MCP. However, exposure to FeSO4-EDTA-ascorbate resulted in little or no dissociation of the complex. The MCP complex dissociated by p-(chloromercuri) benzenesulfonic acid could be reassociated upon treatment with the reducing agent dithiothreitol, but dithiothreitol failed to completely reassociate 3,4-dichloroisocoumarin- or H2O2 treated MCP. Therefore, chemical modification of the MCP can cause activation with varying degrees of complex dissociation. These results suggest that metabolites, such as reactive oxygen species, in addition to endogenous proteins, such as PA28, are capable of modulating MCP activity.
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PMID:Activation of the multicatalytic endopeptidase by oxidants. Effects on enzyme structure. 867 41

The aim of the present study was to characterize human CYP2E1 turnover and examine the possible proteolytic pathways responsible for the rapid degradation of CYP2E1 in a transfected HepG2 cell line expressing human CYP2E1. Two methods were used to study the CYP2E1 turnover; after addition of cycloheximide, the half-life of the CYP2E1 in the intact cells was about 6 h as detected by PNP catalytic activity assay and immunoblot analysis of apoprotein content. CYP2E1 substrates or ligands such as 4-methylpyrazole, ethanol, glycerol, and dimethyl sulfoxide protected CYP2E1 against this rapid degradation, whereas CCl4 accelerated this process. The second procedure involved pulse-chase experiments after labeling CYP2E1 with [35S]methionine and immunoprecipitation with anti-human CYP2E1 IgG. The half-life of CYP2E1 was about 2.5 h, and the various substrates or ligands modified the turnover process within intact cells as described for the cycloheximide experiments. More than 20 different reagents including antioxidants, physiological metabolites, lysosomal inhibitors, and protease inhibitors were screened for possible effects on CYP2E1 proteolytic degradation. Dibutyryl cAMP had no effect on CYP2E1 activity or turnover. Among those reagents tested so far, the serine protease inhibitor 1-chloro-3-tosylamido-7-amino-2-heptanone hydrochloride exhibited some protection against CYP2E1 degradation. To demonstrate whether the proteasome complex is involved in this process, Czb-Ile-Glu(OtBu)-Ala-leucinal (PSI) as a cell penetrating aldehydic proteasome inhibitor and Czb-Leu-norleucinal (calpeptin inhibitor) as an aldehydic nonproteosomal protease inhibitor were used to examine their effect on both the normal and the CCl4-stimulated CYP2E1 proteolytic degradation pathways. Treatment with PSI at concentrations ranging from 5 to 80 microM resulted in a dose-dependent protection against the loss of both the normal CYP2E1 and the CCl4-modified CYP2E1. The maximum protection by PSI at a concentration of 80 microM after a 12-h chase period was about 60% in cells treated with 2 mM CCl4 or 75% in cells without CCl4 treatment. Calpeptin inhibitor afforded little or no protection against CYP2E1 degradation in the absence or presence of CCl4. PSI did not inhibit CYP2E1 catalytic activity, suggesting that it was not a ligand for CYP2E1. These results indicate that human CYP2E1 has a short half-life span and that substrates can significantly modify its turnover rate in intact HepG2 cells. The proteasome proteolytic pathway may be involved in the degradation process of both the normal and the CCl4-modified human CYP2E1 in this model.
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PMID:Characterization of cytochrome P4502E1 turnover in transfected HepG2 cells expressing human CYP2E1. 914 49

Interleukin-1beta (IL-1beta) has been implicated as an effector molecule of beta-cell destruction in autoimmune diabetes. IL-1beta inhibits insulin secretion from pancreatic beta-cells by stimulating the expression of inducible nitric oxide synthase (iNOS) that generates the free radical nitric oxide. IL-1beta also induces the coexpression of the inducible isoform of cyclooxygenase (COX-2) that results in the overproduction of proinflammatory prostaglandins. The current studies were designed to characterize the involvement of protease(s) in the signaling pathway of IL-1beta-induced iNOS and COX-2 expression by rat islets and transformed rat pancreatic beta-cells. Because of the limitations of cell numbers of purified primary beta-cells obtained from rat islets, biochemical and molecular studies were performed using the rat insulinoma beta-cell line RINm5F. A serine protease inhibitor, Nalpha-P-tosyl-L-lysine chloromethyl ketone (TLCK), and a proteasome complex (26S) inhibitor, MG 132, inhibited IL-1beta-induced nitrite formation, an oxidation product of nitric oxide produced by iNOS, in a concentration-dependent manner, with complete inhibition observed at 100 micromol/l and 10 micromol/l, respectively. Both TLCK and MG 132 also inhibited iNOS gene expression at the level of mRNA and protein. In an analogous manner, TLCK (100 micromol/l) and MG 132 (10 micromol/l) inhibited IL-1beta-induced COX-2 enzyme activity (PGE2 formation) and COX-2 gene expression at the level of mRNA and protein. In human islets, the proteasome inhibitor MG 132 also inhibited the formation of the products of iNOS and COX-2 enzyme activity, nitrite, and PGE2, respectively. These findings suggest that the inhibitory action of TLCK and MG 132 on iNOS and COX-2 expression precedes transcription. The transcription factor NFkappaB is essential for activation of a number of cytokine-inducible enzymes and was evaluated as a possible site of protease action necessary for IL-1beta-induced coexpression of iNOS and COX-2. TLCK and MG 132 inhibited both IL-1beta-induced activation of NFkappaB and degradation of IkappaBalpha by islets and RINm5F cells. These results implicate protease activation as an early signaling event in IL-1beta-induced inhibition of beta-cell function. This study also suggests that IL-1beta-induced iNOS and COX-2 coexpression by pancreatic beta-cells share a common signaling pathway in utilizing the proteasome complex (26S) and the transcription factor NFkappaB, and it identifies sites of intervention to prevent the overproduction of their inflammatory products.
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PMID:Evidence for involvement of the proteasome complex (26S) and NFkappaB in IL-1beta-induced nitric oxide and prostaglandin production by rat islets and RINm5F cells. 956 91

Apolipoprotein B100 (apoB) is a large secretory protein that forms very low density lipoprotein in liver. An in vitro degradation assay was developed using rabbit reticulocyte (RR) lysate in order to investigate the mechanism of intracellular degradation of newly synthesized apoB by the ubiquitin-proteasome pathway. [3H]apoB, isolated from [3H]leucine pulsed/chased Hep G2 cells, was degraded 51% when incubated for 2 h at 37 degreesC in an assay mixture that included RR lysate (source of the ubiquitin conjugation system and proteasome) and an exogenous ATP regenerating system. ApoB degradation was ATP-dependent and degradation fragments were not observed suggesting that the very large apoB molecule was extensively degraded. ApoB degradation was decreased to 50% when potent proteasome inhibitors, clasto-lactacystin beta-lactone (10 microM) or MG-132 (50 microM), were added to the reaction mixture, but was not affected by the cysteine protease inhibitor, E-64, or the serine protease inhibitor, phenylmethylsulfonyl fluoride. ApoB degradation was inhibited by the mutant ubiquitin protein K48R and by ubiquitin aldehyde, an inhibitor of ubiquitin-protein isopeptidases. During incubation ubiquitination of apoB increased even as apoB was being degraded. These results suggest that in vitro degradation of apoB, a large secretory protein that is normally found in the endoplasmic reticulum (ER) lumen or associated with the ER membrane, was proteasome-dependent and involved both ubiquitination and deubiquitination steps.
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PMID:Ubiquitin-proteasome-dependent degradation of apolipoprotein B100 in vitro. 993 44

The retinoblastoma protein plays a critical role in regulating the G1/S transition. Less is known about the function and regulation of the homologous pocket protein p107. Here we present evidence for the posttranslational regulation of p107 by the Ca2+-activated protease calpain. Three negative growth regulators, the HMG-CoA reductase inhibitor lovastatin, the antimetabolite 5-fluorouracil, and the cyclic nucleotide dibutyryl cAMP were found to induce cell type-specific loss of p107 protein which was reversible by the calpain inhibitor leucyl-leucyl-norleucinal but not by the serine protease inhibitor phenylmethylsulfonylfluoride, caspase inhibitors, or lactacystin, a specific inhibitor of the 26S proteasome. Purified calpain induced Ca2+-dependent p107 degradation in cell lysates. Transient expression of the specific calpain inhibitor calpastatin blocked the loss of p107 protein in lovastatin-treated cells, and the half-life of p107 was markedly lengthened in lovastatian-treated cells stably transfected with a calpastatin expression vector versus cells transfected with vector alone. The data presented here demonstrate down-regulation of p107 protein in response to various antiproliferative signals, and implicate calpain in p107 posttranslational regulation.
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PMID:Posttranslational regulation of the retinoblastoma gene family member p107 by calpain protease. 1008 33

In the human hepatic cell line, HepG2, apolipoprotein B100 (apoB100) degradation is increased by inhibiting lipid transfer mediated by the microsomal triglyceride transfer protein (MTP) and is predominantly accomplished by the ubiquitin-proteasome pathway. In the current study, we determined whether this degradative pathway was restricted to HepG2 cells or was of more general importance in hepatic apoB100 metabolism. Rat hepatoma McArdle RH7777 cells (McA), compared to HepG2 cells, secrete a large fraction of apoB100 associated with VLDL particles, as does the normal mammalian liver. In McA cells studied under basal conditions, the proteasome inhibitor lactacystin (LAC) increased apoB100 recovery, indicating that the role of the proteasome in apoB100 metabolism is not restricted to HepG2 cells. When apoB100 lipidation was blocked by an inhibitor of MTP (MTPI), recovery of cellular apoB100 was markedly reduced, but LAC was only partially ( approximately 50%) effective in reversing the induced degradation. This partial effectiveness of LAC may have represented either (1) incomplete inhibition by LAC of its preferred target, the chymotrypsin-like activity of the proteasome, (2) the presence of an apoB100 proteolytic activity of the proteasome resistant to LAC, or (3) a nonproteasomal proteolytic pathway of apoB100 degradation. By studying immunoisolated proteasomes and McA cells treated with LAC and/or MTPI and a variety of protease inhibitors, we determined that the proteasomal component of apoB100 degradation was entirely attributable to the chymotrypsin-like catalytic activity, but only accounted for part of apoB100 degradation induced by MTPI. The nonproteasomal apoB100 degradative pathway was nonlysosomal and resistant to E64d, DTT, and peptide aldehydes such as MG132 or ALLN but was partially sensitive to the serine protease inhibitor APMSF. Furthermore, when the protein trafficking inhibitor, brefeldin A, was used to block endoplasmic reticulum (ER) to Golgi transport in MTPI-treated McA cells, degradative activity resistant to LAC was increased, suggesting that the nonproteasomal pathway is associated with the ER.
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PMID:The inhibition of microsomal triglyceride transfer protein activity in rat hepatoma cells promotes proteasomal and nonproteasomal degradation of apoprotein b100. 1214 75


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