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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)

A sequence motif complementary to the nuclear localization signal (NLS) has been localized in proteasomes from Thermoplasma acidophilum by immunoelectron microscopy using sequence-specific antibodies. The antibodies were generated in two different ways: by immunization with a carrier-coupled peptide and by isolation of the sequence-specific antibody from an immune serum against native proteasomes using a peptide-affinity column. The sequence specificity of the isolated antibody was confirmed by a PEPSCAN-ELISA performed on overlapping nonapeptides deduced from the sequence of the alpha-subunit of the Thermoplasma proteasome. Compared to the antibody induced by the carrier-coupled peptide this antibody fraction showed a much higher affinity for native proteasomes. The attachment site of the Fab portion of the antibody to the proteasome was mapped by electron microscopy in conjunction with image processing. The antibody was found to bind to the periphery of the two outer "disks" of the proteasome complex formed by the alpha-subunits.
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PMID:Localization of a sequence motif complementary to the nuclear localization signal in proteasomes from Thermoplasma acidophilum by immunoelectron microscopy. 128 18

Major histocompatibility complex (MHC) class I molecules bind and deliver peptides derived from endogenously synthesized proteins to the cell surface for survey by cytotoxic T lymphocytes. It is believed that endogenous antigens are generally degraded in the cytosol, the resulting peptides being translocated into the endoplasmic reticulum where they bind to MHC class I molecules. Transporters containing an ATP-binding cassette encoded by the MHC class II region seem to be responsible for this transport. Genes coding for two subunits of the '20S' proteasome (a multicatalytic proteinase) have been found in the vicinity of the two transporter genes in the MHC class II region, indicating that the proteasome could be the unknown proteolytic entity in the cytosol involved in the generation of MHC class I-binding peptides. By introducing rat genes encoding the MHC-linked transporters into a human cell line lacking both transporter and proteasome subunit genes, we show here that the MHC-encoded proteasome subunit are not essential for stable MHC class I surface expression, or for processing and presentation of antigenic peptides from influenza virus and an intracellular protein.
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PMID:Proteasome subunits encoded by the major histocompatibility complex are not essential for antigen presentation. 129 22

Two-types of high molecular mass proteases have been purified from sea urchin sperm using DEAE-Sephacel, hydroxylapatite and Superdex 200 column chromatography. Both proteases showed similar hydrolyzing activities toward synthetic peptides, but they differed in the molecular mass and peptide composition. One was probably identical to a proteasome (multicatalytic proteinase), judging from its molecular mass (650 kDa) and polypeptide composition. The other one was composed of several polypeptides with molecular masses ranging from 24 kDa to 125 kDa and its molecular mass was estimated as 950 kDa by gel filtration. These two proteases, however, were closely related to each other. Immunological studies revealed that the 950-kDa protease comprised at least five subunits of the 650-kDa protease.
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PMID:Two high molecular mass proteases from sea urchin sperm. 131 Mar 90

An inhibitory protein for the 20S proteasome (also known as macropain, the multicatalytic proteinase complex and 20S proteinase) has been purified from bovine red blood cells. The inhibitor has an apparent molecular weight of 31,000 on SDS-PAGE and appears to form multimers under nondenaturing conditions. This protein inhibited all three of the putatively distinct catalytic activities of proteasome A (the active form of the proteinase) characterized by the hydrolysis of synthetic peptides such as Z-VLR-MNA, Z-GGL-AMC or Suc-LLVY-AMC and Z-LLE-beta NA. The inhibitor also prevented the hydrolysis of large protein substrates such as casein, lysozyme and bovine serum albumin. Proteasome L (the latent form of the proteinase) does not degrade these large protein substrates, but does hydrolyze the three synthetic peptides at rates similar to those by proteasome A. The inhibitor inhibited only two of these peptidase activities of proteasome L (hydrolysis of Z-GGL-AMC and of Z-LLE-beta NA or Suc-LLVY-AMC); it had no effect on the hydrolysis of Z-VLR-MNA. The inhibitor was specific for inhibition of the proteasome and had no effect on the activity of any other proteinase tested including trypsin, chymotrypsin, papain, subtilisin and both isoforms of calpain. Kinetic analysis indicates that the inhibitor interacted with the proteasome by a mechanism involving tight-binding. Because the proteasome appears to be a key component of the ATP/ubiquitin-dependent pathway of intracellular protein degradation, the inhibitor may represent an important regulatory protein of this pathway.
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PMID:Purification and characterization of a protein inhibitor of the 20S proteasome (macropain). 131 59

Proteins conjugated to ubiquitin are degraded by a 26S (1500-kDa) proteolytic complex that, in reticulocyte extracts, can be formed by the association of three factors: CF-1, CF-2, and CF-3. One of these factors, CF-3, has been shown to be the proteasome, a 650-kDa multicatalytic protease complex. We have purified a 250-kDa inhibitor of the proteasome and shown that it corresponds to CF-2. In the presence or absence of ATP, this factor inhibited hydrolysis by the proteasome of both fluorogenic tetrapeptides and protein substrates. When the inhibitor, proteasome, and CF-1 were incubated together in the presence of ATP and Mg2+, degradation of ubiquitin-125I-lysozyme occurred. Both the inhibitory activity and the ability to reconstitute ubiquitin-125I-lysozyme degradation were very labile at 42 degrees C, but both activities were stabilized by ATP or a nonhydrolyzable ATP analog. SDS/PAGE indicated that the 250-kDa inhibitor fraction contained a major subunit of 40 kDa (plus some minor bands). The 125I-labeled inhibitor and purified proteasome formed a complex. When CF-1, ATP, and Mg2+ were also present, the 125I-labeled inhibitor along with the proteasome formed a complex of 1500 kDa. The inhibitor (CF-2) thus appears to be an ATP-binding component that regulates proteolysis within the 1500-kDa complex.
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PMID:An ATP-stabilized inhibitor of the proteasome is a component of the 1500-kDa ubiquitin conjugate-degrading complex. 131 79

It is known that two types of high-molecular-mass protease complexes are present in the cytosol of mammalian cells; a 20S latent multicatalytic proteinase named the proteasome, and a large proteolytic complex with an apparent sedimentation coefficient of 26S that catalyzes ATP-dependent breakdown of proteins conjugated with ubiquitin. In this work, we first demonstrated that a low concentration of SDS was required for activation of the latent proteasome, whereas the 26S complex degraded substrates for proteasomes in the absence of SDS. Moreover, the 26S complex was greatly stabilized in the presence of 2 mM ATP and 20% glycerol. Based on these characteristics, we next devised a novel procedure for purification of the 26S proteolytic complexes from human kidney. In this procedure, the proteolytic complexes were precipitated from cytoplasmic extracts by ultracentrifugation for 5 h at 105000 x g, and the large 26S complexes were clearly separated from the 20S proteasomes by molecular-sieve chromatography on a Biogel A-1.5 m column. The 26S enzyme was then purified to apparent homogeneity by successive chromatographies on hydroxyapatite and Q Sepharose, then by glycerol density-gradient centrifugation. Electrophoretic and immunochemical analyses showed that the purified human 26S complex consisted of multiple subunits of proteasomes with molecular masses of 21-31 kDa and 13-15 protein components ranging in molecular mass over 35-110 kDa, which were directly associated with the proteasome. The purified 26S proteolytic complex degraded 125I-labeled lysozyme-ubiquitin conjugates in an ATP-dependent manner. The 26S enzyme also showed high ATPase activity, which was copurified with the complex. Vanadate and hemin strongly inhibited not only ATP cleavage, but also ATP-dependent breakdown of ubiquitinligated proteins, suggesting that the 26S complex hydrolyzes ATP and ubiquitinated proteins by closely linked mechanisms. These findings indicate that the 26S complex consists of a proteasome with proteolytic function and multiple other components including an ATPase that regulates energy-dependent, ubiquitin-mediated protein degradation.
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PMID:Demonstration that a human 26S proteolytic complex consists of a proteasome and multiple associated protein components and hydrolyzes ATP and ubiquitin-ligated proteins by closely linked mechanisms. 131 98

Proteinase yscE, the proteasome/multicatalytic-multifunctional proteinase of yeast had been shown to function in stress response and in the degradation of ubiquitinated proteins [(1991) EMBO J. 10, 555-562]. A well-defined set of proteins degraded via ubiquitin-mediated proteolysis are the substrates of the N-end rule pathway [(1986) Science 234, 179-186; (1989) Science 243, 1576-1583]. We show that mutants defective in the chymotryptic activity of proteinase yscE fail to degrade substrates of the N-end rule pathway. This gives further proof of the proteasome being a central catalyst in ubiquitin-mediated proteolysis.
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PMID:The proteasome/multicatalytic-multifunctional proteinase. In vivo function in the ubiquitin-dependent N-end rule pathway of protein degradation in eukaryotes. 132 27

A major eukaryotic proteolytic system is known to require the covalent attachment of ubiquitin to substrates prior to their degradation, yet the proteinase involved remains poorly defined. The proteasome, a large conserved multi-subunit protein complex of the cytosol and the nucleus, has been implicated in a variety of cellular functions. It is shown here that a yeast mutant with a defective proteasome fails to degrade proteins which are subject to ubiquitin-dependent proteolysis in wild-type cells. Thus, the proteasome is part of the ubiquitin system and mediates the degradation of ubiquitin-protein conjugates in vivo.
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PMID:In vivo function of the proteasome in the ubiquitin pathway. 132 95

The multicatalytic proteinase (proteasome; MCP) is a high molecular mass proteinase which is found in all eukaryotic cells. Northern blot analysis of the levels of MCP mRNAs in a Rat-1 fibroblast cell line and in cells transformed with Rous sarcoma virus showed marked increases in the transformed cells. However, the results of immunoblot analysis with anti-MCP antibodies suggested that the MCP protein content of the two cell lines was similar.
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PMID:Enhanced levels of multicatalytic proteinase mRNAs in Rous sarcoma virus transformed cells. 132 57

Western blot analysis, using a polyclonal antibody to the 240-kDa endogenous inhibitor of the 20 S proteasome, revealed that the inhibitor is a component of the 26 S complex. Although isolated inhibitor displayed a single 40-kDa band on SDS-PAGE, the antibody detected a 55-kDa component in the 26 S proteasome complex. Ubiquitin polyclonal antibody recognized the same 55-kDa component but did not react with free 40-kDa inhibitor subunit. Addition of purified 40-kDa inhibitor to a ubiquitin ligating system also generated the 55-kDa species. In crude erythrocyte extracts, most of the inhibitor migrated at 55 kDa in the presence of ATP but shifted to 40 kDa in the absence of ATP, consistent with removal of ubiquitin. It is suggested that ubiquitination of the inhibitor may be involved in regulating assembly and/or activity of the 26 S proteasome complex.
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PMID:Ubiquitinated proteasome inhibitor is a component of the 26 S proteasome complex. 133 90


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