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)

Lactacystin is a Streptomyces metabolite that inhibits cell cycle progression and induces neurite outgrowth in a murine neuroblastoma cell line. Tritium-labeled lactacystin was used to identify the 20S proteasome as its specific cellular target. Three distinct peptidase activities of this enzyme complex (trypsin-like, chymotrypsin-like, and peptidylglutamyl-peptide hydrolyzing activities) were inhibited by lactacystin, the first two irreversibly and all at different rates. None of five other proteases were inhibited, and the ability of lactacystin analogs to inhibit cell cycle progression and induce neurite outgrowth correlated with their ability to inhibit the proteasome. Lactacystin appears to modify covalently the highly conserved amino-terminal threonine of the mammalian proteasome subunit X (also called MB1), a close homolog of the LMP7 proteasome subunit encoded by the major histocompatibility complex. This threonine residue may therefore have a catalytic role, and subunit X/MB1 may be a core component of an amino-terminal-threonine protease activity of the proteasome.
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PMID:Inhibition of proteasome activities and subunit-specific amino-terminal threonine modification by lactacystin. 773 82

Two high molecular mass proteinases, multicatalytic proteinase (MCP) and a new high molecular mass proteinase (HMP) with only chymotrypsin-like activity (Khan et al. (1994) J. Biol. Chem. 269, 10016-10021) from human erythrocyte membranes, have been compared. For this purpose, MCP was purified from human erythrocyte membranes in the active form towards synthetic peptide substrates; it also hydrolysed the protein substrates [14]methyl casein and [14C]oxidised insulin beta chain at 37 degrees C. MCP from plasma membranes exhibited hollow cylindrical structures also typical of cytosolic forms. Radiolabelled diisopropyl fluorophosphate, [3H]DFP, a serine proteinase inhibitor, labelled a band of Mr 23 000 in membrane MCP. By contrast, no labelling was obtained with HMP. Chymotrypsin-like activity of HMP was also found to be insensitive to DFP. On the other hand, DFP inhibited chymotrypsin-like and peptidylglutamyl peptide hydrolysing activities of membrane MCP, with no effect on its trypsin-like activity. The inhibition of MCP by DFP was concentration-dependent. These studies showed that MCP and HMP represent two distinct kinds of proteinases with chymotrypsin-like activities and can be distinguished by the serine proteinase inhibitor DFP.
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PMID:Membrane-bound high molecular mass proteinases from human erythrocytes. 781 93

The multicatalytic proteinase (MCP or proteasome) is a large proteolytic complex that contains at least five catalytic components: the trypsin-like, chymotrypsin-like, peptidylglutamyl-peptide hydrolase (PGPH), branched-chain-amino-acid-preferring (BrAAP) and small-neutral-amino-acid-preferring activities. We have shown that brief heating of the lobster muscle proteasome activates a proteolytic activity that degrades casein and myofibrillar proteins and is distinct from the trypsin-like, chymotrypsin-like and PGPH components. Here we identify the BrAAP activity as a catalytic component involved in the initial degradation of myofibrillar proteins in vitro. This conclusion is based on the following. (1) The BrAAP component was activated by heat-treatment, whereas the other four peptidase activities were not. (2) The BrAAP and proteolytic activities showed similar sensitivities to cations and protease inhibitors: both were inhibited by 3,4-dichloroisocoumarin, chymostatin, N-ethylmaleimide and Mg2+, but were not affected by leupeptin, phenylmethanesulphonyl fluoride or Li+. (3) The BrAAP activity was inhibited most strongly by casein substrates and troponin; conversely, the troponin-degrading activity was inhibited by the BrAAP substrate. Another significant finding was that incubation of the heat-activated MCP in the presence of chymostatin resulted in the limited cleavage of troponin-T2 (45 kDa) to two fragments of 41 and 42 kDa; this cleavage was completely suppressed by leupeptin. These results suggest that under certain conditions the trypsin-like component can cleave endogenous protein.
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PMID:Branched-chain-amino-acid-preferring peptidase activity of the lobster multicatalytic proteinase (proteasome) and the degradation of myofibrillar proteins. 786 22

The multicatalytic proteinase complex (MPC) or proteasome is a multimeric, high-molecular-weight (700,000), extralysosomal proteolytic enzyme found in eukaryotes and in archaebacteria. Its multiple catalytic sites grant it a broad cleavage specificity toward short peptides and protein substrates. The pH optima of the catalytic activities of MPC are in the neutral or slightly alkaline range. We present here evidence for cryptic catalytic components of MPC optimally active at an acidic pH. Studies with a hydrophobic fluorescent probe provide direct evidence for conformational changes brought about by exposing the complex to an acidic environment. One of the newly described components, designated "acidic chymotrypsin-like activity," cleaves the Leu-2-naphthylamide bond in the substrate Boc-Val-Glu-Ala-Leu-2-naphythylamide. Compared with the classical "neutral" chymotrypsin-like activity defined by cleavage of the Leu-p-nitroanilide bond in Z-Gly-Gly-Leu-p-nitroanilide, the newly described component is not inhibited by monovalent cations and is less sensitive to the peptidyl aldehyde Z-Gly-Gly-leucinal, an inhibitor of the neutral chymotrypsin-like activity. In addition, we describe the properties of a novel potent peptidyl aldehyde, Z-Ile-Glu(OtBu)-Ala-leucinal, which is an inhibitor of both the acidic and neutral chymotrypsin-like activities of MPC, with IC50 values of 0.25 and 6.5 microM, respectively. In the presence of 65 microM of the newly synthesized peptidyl aldehyde, other MPC components such as the trypsin-like and peptidyl-glutamyl peptide hydrolyzing activities were decreased only by 14 and 9%, respectively. The hydrophobicity, potency, and specificity of Z-Ile-Glu(OtBu)-Ala-leucinal toward the chymotrypsin-like activities of the complex make it a valuable pharmacological tool with which to investigate the physiological roles of MPC.
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PMID:A novel chymotrypsin-like component of the multicatalytic proteinase complex optimally active at acidic pH. 787 5

Exposure of HT4 cells (a mouse neuronal cell line) to a new potent permeable peptidyl aldehyde inhibitor of the chymotrypsin-like activity of the multicatalytic proteinase complex (MPC) causes accumulation of ubiquitinylated proteins. In contrast, inhibition of calpain or treatment with a lysosomotropic agent failed to produce detectable ubiquitin-protein conjugates. The appearance of such conjugates is not a nonspecific phenomenon because incubation with the peptidyl alcohol analogue of the inhibitor does not produce accumulation of ubiquitinylated proteins. The MPC inhibitor may therefore be a useful tool for identification and study of physiological pathways involving MPC. Furthermore, the inhibitor may help develop a model for the study of neurodegeneration where accumulation of ubiquitin-protein conjugates is commonly detected in abnormal brain inclusions.
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PMID:A new inhibitor of the chymotrypsin-like activity of the multicatalytic proteinase complex (20S proteasome) induces accumulation of ubiquitin-protein conjugates in a neuronal cell. 793 14

One of the main components of the senile plaques in brain tissue from patients with Alzheimer's disease is the beta-amyloid peptide. This peptide is proteolytically cleaved from the amyloid precursor protein by the action of at least two proteases, a beta-secretase which generates the N-terminus and a gamma-secretase which generates the C-terminus. Neither of these proteases have been identified. To identify proteases that are candidates for the gamma-secretase we synthesized a small fluorescent peptide substrate containing the amino acids comprising the C-terminus of the longest beta-amyloid peptide identified. This substrate is hydrolyzed by a single activity in homogenates from both cells and brain tissue and we have demonstrated that this activity is the multicatalytic enzyme or proteasome. Furthermore, using specific inhibitors, we have demonstrated that the fluorescent substrate is hydrolyzed by the chymotrypsin-like activity of the multicatalytic enzyme.
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PMID:Identification of the multicatalytic enzyme as a possible gamma-secretase for the amyloid precursor protein. 794 79

Activation of the inducible transcription factor NF-kappa B involves removal of the inhibitory subunit I kappa B-alpha from a latent cytoplasmic complex. It has been reported that I kappa B-alpha is subject to both phosphorylation and proteolysis in the process of NF-kappa B activation. In this study, we present evidence that the multicatalytic cytosolic protease (proteasome) is involved in the degradation of I kappa B-alpha. Micromolar amounts of the peptide Cbz-Ile-Glu(O-t-Bu)-Ala-leucinal (PSI), a specific inhibitor of the chymotrypsin-like activity of the proteasome, prevented activation of NF-kappa B in response to tumor necrosis factor-alpha (TNF) and okadaic acid (OA) through inhibition of I kappa B-alpha degradation. The m-calpain inhibitor Cbz-Leu-leucinal was ineffective. In the presence of PSI, a newly phosphorylated form of I kappa B-alpha accumulated in TNF- and OA-stimulated cells. However, the covalent modification of I kappa B-alpha was not sufficient for activation of NF-kappa B: no substantial NF-kappa B DNA binding activity appeared in cells because the newly phosphorylated form of I kappa B-alpha was still tightly bound to p65 NF-kappa B. Pyrrolidinedithiocarbamate, an antioxidant inhibitor of NF-kappa B activation which did not interfere with proteasome activities, prevented de novo phosphorylation of I kappa B-alpha as well as its subsequent degradation. This suggests that phosphorylation of I kappa B-alpha is equally necessary for the activation of NF-kappa B.(ABSTRACT TRUNCATED AT 250 WORDS)
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PMID:A proteasome inhibitor prevents activation of NF-kappa B and stabilizes a newly phosphorylated form of I kappa B-alpha that is still bound to NF-kappa B. 795 9

Evidence indicates that a component of the multicatalytic proteinase complex (MPC) that preferentially cleaves bonds after branched chain amino acids (BrAAP) is a major factor responsible for the protein-degrading activity of the MPC. We report here the synthesis of substrate-related peptidyl aldehydes that inhibit the activity of this component toward both synthetic peptide substrates and proteins. The most potent of the inhibitors, Cbz-Gly-Pro-Phe-leucinal (Cbz-GPFL-CHO) inhibits competitively with a Ki of 1.5 microM. The peptidyl aldehydes also inhibit the small neutral amino acid preferring and the peptidylglutamyl-peptide hydrolyzing activities of the MPC. The chymotrypsin-like activity is only weakly inhibited, and the trypsin-like activity is moderately activated. The importance of a Pro residue in the P3 position and a leucinal residue in the P1 position for inhibition of the BrAAP component is indicated by the finding that replacement of these residues by a glycine or phenylalaninal, respectively, markedly increases the Ki. Cbz-GPFL-CHO inhibited the BrAAP activity with the same Ki both before and after activation of this component by exposure of the MPC to 3,4-dichloroisocoumarin, suggesting that the peptidyl aldehyde is an effective inhibitor of both the overt and latent proteolytic activities of the MPC. Incubation of a human breast cancer cell line (MCF-7) in culture with the inhibitors of the BrAAP component led to an accumulation of ubiquitin-protein conjugates, indicating inhibition of the ubiquitin-dependent proteolytic pathway.
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PMID:Inhibition of the proteolytic activity of the multicatalytic proteinase complex (proteasome) by substrate-related peptidyl aldehydes. 796 80

We have recently described nongranular, cytosolic, high-molecular-weight trypsin-like (A-NKP 1) and chymotrypsin-like (A-NKP 2) proteases of interleukin-2-activated rat natural killer (A-NK) cells. A functional correlation between the inactivation of A-NKP 2 and the inhibition of rat A-NK cell-mediated cytotoxicity was found. Herein we describe the 6,000-fold purification of A-NKP 2 to apparent homogeneity following: isopycnic sucrose gradient fractionation of postnuclear supernatants, molecular sieve chromatography, and heparin-Sepharose chromatography. We also report the novel finding that A-NKP 2 as well as A-NKP 1, derived from either rat A-NK cells or the rat NK leukemic cell line CRNK-16, are constituents of the multicatalytic proteinase (MCP/proteasome) complexes of these cells. Characteristic biochemical, biophysical, and electron microscopic/ultrastructural similarity to the rat liver proteasome was observed. However, Western blot analysis using polyclonal antibodies to the rat liver proteasome clearly indicated differences in the rat hepatic proteasome and the CRNK-16-derived proteasomal subunits. The identification, characterization, and purification of A-NKP 1 and A-NKP 2, described herein, now allow for further investigation of the potential role of these proteasome components in NK cell function. Moreover, the proteasome of NK and A-NK cells can now be compared and contrasted to the granzymes of lytic granules with respect to their role in cell-mediated cytotoxicity.
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PMID:Nongranular proteolytic enzymes of rat IL-2-activated natural killer cells. II. Purification and identification of rat A-NKP 1 and A-NKP 2 as constituents of the multicatalytic proteinase (proteasome) complex. 808 94

The proteasome is a 700-kD multisubunit enzyme complex with several proteolytically active sites. The enzyme complex is involved in both ubiquitin-dependent and -independent protein degradation and may contribute to the processing of antigens presented by major histocompatibility complex (MHC) class I molecules. Here we demonstrate that treatment of mouse fibroblast cells with 20 U interferon gamma (IFN-gamma) for 3 d induces a change in the proteasome subunit composition and that the beta-type subunit LMP2, which is encoded in the MHC class II region, is incorporated into the enzyme complex. This is paralleled by reduction of the homologous delta-subunit. IFN-gamma stimulation results in a downregulation of the chymotrypsin-like Suc-LLVY-MCA peptide hydrolyzing activity of 20S proteasomes whereas the trypsin-like activity remains unaffected. When tested as a substrate a synthetic 25-mer polypeptide whose sequence covers the antigenic nonapeptide YPHFMPTNL of the MCMV pp89, 20S proteasomes of IFN-gamma-induced cells exhibit altered chymotrypsin-like cleavage site preferences. In the absence of IFN-gamma induction, the naturally processed nonamer peptide that is presented by MHC class I molecules appears as a minor cleavage product. IFN-gamma activation does not result in an increase of the final peptide but results in a different set of peptides. We hypothesize that these peptides represent precursor peptides that can be trimmed to final peptide size.
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PMID:Interferon gamma stimulation modulates the proteolytic activity and cleavage site preference of 20S mouse proteasomes. 811 82


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