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 (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

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

The biochemical properties of heretofore uncharacterized extracellular proteases of rat interleukin-2-activated natural killer (A-NK) cells were investigated. Following p-aminobenzamidine- and D-phenylalanine-agarose affinity chromatography both trypsin- and chymotrypsin-like protease activities were found. Zymography revealed "trypsin-like" activities, with apparent molecular weights of approximately 42 kDa to 20.5 kDa, and "chymotrypsin-like" activities of approximately 27 kDa, to 22 kDa. These proteases might contribute to diverse NK cell functions, and the characteristics of these proteolytic activities are compared with those of granzymes of lytic granules and the proteasome of A-NK cells.
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PMID:Zymographic analysis of extracellular, released and cell-associated proteases of rat interleukin 2-activated natural killer (A-NK) cells. 805 9

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

We previously identified a benzyloxycarbonyl(Z)-Leu-Leu-Leu-4-methylcoumaryl-7-amide (ZLLL-MCA) degrading activity in proteasome as a candidate for the regulator of neurite outgrowth. As its counterpart, we purified a protein from bovine brain that specifically inhibits the ZLLL-MCA degrading activity in proteasome. This protein is heat stable and has no effect on the other catalytic activities in proteasome, or on the activities of trypsin, chymotrypsin, or m- and mu-calpains either. The molar ratio of inhibitor-to-proteasome that inhibits 50% of the ZLLL-MCA degrading activity of proteasome is 1:1. The inhibitory mechanism of the inhibitor against proteasome is non-competitive. Finally, the inhibitor was identified as heat-shock protein 90 (HSP90) by partial amino acid sequencing and immunodetection. The results suggest that HSP90 initiates neurite outgrowth through the inhibition of the ZLLL-MCA degrading activity in proteasome.
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PMID:Purification and characterization of an endogenous inhibitor specific to the Z-Leu-Leu-Leu-MCA degrading activity in proteasome and its identification as heat-shock protein 90. 818 90

To obtain information on the role of proteasomes in the immune system, we examined the effect of a major immunomodulatory cytokine, gamma interferon (IFN-gamma), on the expressions, structures, and functions of proteasomes. IFN-gamma greatly increased the levels of the mRNAs encoding LMP2 and LMP7, putative immuno-proteasome subunits encoded by genes within the class II MHC region, and these two subunits synthesized were assembled completely into the proteasomal multi-subunit complex in various types of human cells. The subunit organization of proteasome changed in response to IFN-gamma stimulation, due to assembly of newly synthesized subunits through up- and down-expressions of at least 6 proteasome genes including LMP2/LMP7 without change in the structure of pre-existing proteasomes. Interestingly, IFN-gamma dramatically stimulated the trypsin-like and chymotrypsin-like activities of the multifunctional proteasome and depressed the peptidylglutamyl-peptide-hydrolyzing activity, without affecting the activity for ATP-, ubiquitin-dependent proteolysis. These results indicate that IFN-gamma modifies not only the structural organization of the proteasome, but also its functions. Based on these findings, we discuss the role in the antigen processing/presentation pathway of proteasomes with functional diversity acquired through alteration of their subunit assembly in response to IFN-gamma stimulation.
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PMID:Interferon-gamma induces different subunit organizations and functional diversity of proteasomes. 820 75

The effect of phospholipids on the trypsin-like, chymotrypsin-like and peptidylglutamyl-peptide-hydrolysing activities of the so-called latent form of the rat liver multicatalytic proteinase was studied, assaying them with the following substrates: N-Cbz-ARR-4MNA (N-Cbz, N-benzyloxycarbonyl; 4MNA, 4-methoxy-beta-naphthylamide), N-Suc-LLVY-MCA (N-Suc, N-succinyl; MCA, methylcoumarin) and N-Cbz-LLE-beta-NA (beta-NA, beta-naphthylamide) respectively (amino acids are shown as their one-letter symbol). For the most part neither lysophospholipids nor phospholipids at 20 micrograms/ml have any effect on the activity of the enzyme (assayed at 50 microM peptide), except for phosphatidylserine, which activates 2-fold the hydrolysis of N-Suc-LLVY-MCA, and phosphatidylinositol, which inhibits by 20% the hydrolysis of N-Cbz-LLE-beta-NA. By contrast, cardiolipin (diphosphatidylglycerol) is a strong activator of the hydrolysis of N-Suc-LLVY-MCA (60-fold) and N-Cbz-LLE-beta-NA (30-fold), with half-maximal activation at concentrations of 0.15 micrograms/ml and 1.5 micrograms/ml respectively. The activation of N-Suc-LLVY-MCA hydrolysis is due to an increase of the affinity of the enzyme for the peptide and to an increase in the Vmax. (30-fold). The activation of N-Cbz-LLE-beta-NA hydrolysis is explained by suppressing the co-operativity for this substrate, producing hyperbolic kinetics with a Km of 60 microM and a 15-fold increase in the Vmax. of the enzyme. This activation by cardiolipin was completely suppressed by micromolar concentrations of fluophenazine, a drug known to inhibit other phospholipid-regulated process. Cardiolipin activation and the known activation by SDS are additive, either at suboptimal or optimal concentrations of both activators. Cardiolipin also activates the in vitro degradation of some proteins from metabolically labelled total cellular extracts by the latent multicatalytic proteinase. These results clearly show that cardiolipin is a natural positive modulator of the peptidase and proteolytic activities of the multicatalytic proteinase, probably acting through a binding site different from that of SDS.
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PMID:Kinetic mechanism of activation by cardiolipin (diphosphatidylglycerol) of the rat liver multicatalytic proteinase. 825 Aug 60

The fate of a mutant form of each of the two yeast vacuolar enzymes proteinase yscA (PrA) and carboxypeptidase yscY (CPY) has been investigated. Both mutant proteins are rapidly degraded after entering the secretory pathway. Mutant PrA is deleted in 37 amino acids spanning the processing site region of the PrA pro-peptide. The mutant enzyme shows no activity towards maturation of itself or other vacuolar hydrolases, a function of wild-type PrA. Mutant CPY carries an Arg instead of a Gly residue in a highly conserved region, two positions distant from the active-site Ser. In contrast to wild-type CPY, the mutant form was quickly degraded by trypsin in vitro, indicating an altered structure. Using antisera specific for alpha-1-->6 and alpha-1-->3 outer-chain mannose linkages, no Golgi-specific carbohydrate modification could be detected on either mutant protein. Subcellular fractionation studies located both mutant enzymes in the endoplasmic reticulum. Degradation kinetics of both proteins show the same characteristics, indicating similar degradation pathways. The degradation process was shown to be independent of a functional sec18 gene product and takes place before Golgi-specific carbohydrate modifications occur. The proteasome, the major proteolytic activity of the cytoplasm, is not involved in this degradation event. All degradation characteristics of the two mutant proteins are consistent with a degradation process within the endoplasmic reticulum ('ER degradation').
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PMID:Analysis of two mutated vacuolar proteins reveals a degradation pathway in the endoplasmic reticulum or a related compartment of yeast. 826 47


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