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)

A major metabolic effect of insulin is inhibition of cellular proteolysis, but the proteolytic systems involved are unclear. Tissues have multiple proteolytic systems, including the ATP- and ubiquitin-dependent proteasome pathway. The effect of insulin on this pathway was examined in vitro and in cultured cells. Insulin inhibited ATP- and ubiquitin-dependent lysozyme degradation more than 90% by reticulocyte extract, in a dose-dependent manner (IC50 approximately 50 nM). Insulin did not reduce the conjugation of ubiquitin to lysozyme and was not itself ubiquitin-conjugated. In HepG2 cells, insulin increased ubiquitin-conjugate accumulation 80%. The association between the 26S proteasome and an intracellular protease, the insulin-degrading enzyme (IDE), was examined by a purification scheme designed to enrich for the 26S proteasome. Copurification of IDE activity and immunoreactivity with the proteasome were detected through several chromatographic steps. Glycerol gradient analysis revealed cosedimentation of IDE with the 20S proteasome and possibly with the 26S proteasome. The proteasome-associated IDE was displaced when the samples were treated with insulin. These results suggest that insulin regulates protein catabolism, at least in part, by decreasing ubiquitin-mediated proteasomal activity, and provides a new target for insulin action. The displacement of IDE from the proteasome provides a mechanism for this insulin action.
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PMID:Insulin inhibits the ubiquitin-dependent degrading activity of the 26S proteasome. 1087 52

The 26S proteasome is a multisubunit protein- destroying machinery that degrades ubiquitin-tagged proteins. To date only a single species of Rpn10, which possibly functions as a multiubiquitin chain-binding subunit, has been identified in various organisms. Here we report that mouse Rpn10 mRNAs occur in at least five distinct forms, named Rpn10a to Rpn10e, and that they are generated from a single gene by developmentally regulated, alternative splicing. Rpn10a is ubiquitously expressed, whereas Rpn10e is expressed only in embryos, with the highest levels of expression in the brain. Both forms of Rpn10 are components of the 26S proteasome, with an apparently similar affinity for multiubiquitylated [(125)I]lysozyme in vitro. However, they exert markedly divergent effects on the destruction of B-type cyclin in Xenopus egg extracts. Thus, the 26S proteasome occurs in at least two functionally distinct forms: one containing a ubiquitously expressed Rpn10a and the other a newly identified, embryo-specific Rpn10e. While the former is thought to perform proteolysis constitutively in a wide variety of cells, the latter may play a specialized role in early embryonic development.
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PMID:Developmentally regulated, alternative splicing of the Rpn10 gene generates multiple forms of 26S proteasomes. 1092 94

Proteasomes, the proteolytic machinery of the ubiquitin/ATP-dependent pathway, have a relevant role in many processes crucial for cell physiology and cell cycle progression. Proteasome inhibitors are used to block cell cycle progression and to induce apoptosis in certain cell lines. Here we examine whether proteasomal function is affected by the anti-tumour drug vinblastine, whose cytostatic action relies mainly on the disruption of mitotic spindle dynamics. The effects of vinblastine on the peptidase activities of human 20 S and 26 S proteasomes and on the proteolytic activity of 26 S proteasome were assessed in the presence of specific fluorogenic peptides and (125)I-lysozyme-ubiquitin conjugates respectively. The assays of ubiquitin-protein conjugates and of inhibitory kappa B alpha (I kappa B alpha), which are characteristic intracellular proteasome substrates, by Western blotting on lysates from HL60 cells incubated with or without vinblastine, illustrated the effects of vinblastine on proteasomes in vivo. We also evaluated the effects of vinblastine on the signal-induced degradation of I kappa B alpha. Vinblastine at 3--110 microM reversibly inhibited the chymotrypsin-like activity of the 20 S proteasome and the trypsin-like and peptidyl-glutamyl-peptide hydrolysing activities of both proteasomes, but only at 110 microM vinblastine was the chymotrypsin-like activity of the 26 S proteasome inhibited; furthermore, at 25--200 microM the drug inhibited the degradation of ubiquitinated lysozyme. In HL60 cells exposed for 6 h to 0.5--10 microM vinblastine, the drug-dose-related accumulation of polyubiquitinated proteins, as well as that of a high-molecular-mass form of I kappa B alpha, occurred. Moreover, vinblastine impaired the signal-induced degradation of I kappa B alpha. Cell viability throughout the test was approx. 95%. Proteasomes can be considered to be a new and additional vinblastine target.
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PMID:Proteasomes are a target of the anti-tumour drug vinblastine. 1138 92

Two distinct activities cleaving bonds after hydrophobic amino acids have been identified in the bovine pituitary 20 S proteasome. One, expressed by the X subunit, that cleaves bonds after aromatic and branched chain amino acids was designated as chymotrypsin-like (ChT-L).(1) The second, expressed by the Y subunit, that cleaves bonds after acidic amino acids was designated as peptidylglutamyl-peptide hydrolyzing (PGPH) but also cleaves bonds after branched chain amino acids. Low micromolar concentrations of the arginine-rich histone H3 (H3) are shown to induce changes in the specificity of the proteasome by selectively activating cleavages after branched chain and acidic amino acids while inhibiting cleavage of peptidyl-arylamide bonds in synthetic substrates. H3 activates 15-fold cleavage after leucine but not phenylalanine residues in model synthetic substrates. The activation is associated with a decrease in K(m) and an increase in V(max), suggesting positive allosteric activation. H3 activates more than 60-fold degradation of the oxidized B-chain of insulin, by cleaving mainly bonds after acidic and branched chain amino acids, and accelerates the degradation of casein and lysozyme, the latter in the presence of dithiothreitol. The degradation of lysozyme in the presence of H3 generates fragments that differ from those in its absence, indicating H3-induced specificity changes. H3 inhibits cleavage of the Trp3-Ser4 and Tyr5-Gly6 bonds in gonadotropin releasing hormone, bonds cleaved by the ChT-L activity in the absence of H3. The results suggest H3-selective activation of the Y subunit and specificity changes that could potentially affect proteasomal function in the nuclear compartment.
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PMID:Selective activation of the 20 S proteasome (multicatalytic proteinase complex) by histone h3. 1173 14

The glycine-alanine repeat (GAr) of the Epstein-Barr virus nuclear antigen-1 is a cis-acting transferable element that inhibits ubiquitin/proteasome-dependent proteolysis in vitro and in vivo. We have here examined the effect of a synthetic 20-mer GAr oligopeptide on the degradation of iodinated or biotin labeled lysozyme in a rabbit reticulocyte lysates in vitro assay. Micromolar concentrations of the GA-20 peptide inhibited the hydrolysis of lysozyme without significant effect on ubiquitination. Addition of the peptide did not inhibit the hydrolysis of fluorogenic substrate by purified proteasomes and did not affect the ubiquitination of lysozyme. An excess of the peptide failed to compete for binding of a synthetic tetra-ubiquitin complex to the S5a ubiquitin-binding subunit of the 19S regulator, confirming that the GAr does not block the access of ubiquitinated substrates to the proteasome. Our data suggest that the GAr may act by destabilizing the interaction of ubiquitinated substrates with the proteasome and promote the premature release of the substrate.
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PMID:Inhibition of ubiquitin-dependent proteolysis by a synthetic glycine-alanine repeat peptide that mimics an inhibitory viral sequence. 1209 25

Oxidatively modified proteins that accumulate in aging and many diseases can form large aggregates because of covalent cross-linking or increased surface hydrophobicity. Unless repaired or removed from cells, these oxidized proteins are often toxic, and threaten cell viability. Most oxidatively damaged proteins appear to undergo selective proteolysis, primarily by the proteasome. Previous work from our laboratory has shown that purified 20 S proteasome degrades oxidized proteins without ATP or ubiquitin in vitro, but there have been no studies to test this mechanism in vivo. The aim of this study was to determine whether ubiquitin conjugation is necessary for the degradation of oxidized proteins in intact cells. We now show that cells with compromised ubiquitin-conjugating activity still preferentially degrade oxidized intracellular proteins, at near normal rates, and this degradation is still inhibited by proteasome inhibitors. We also show that progressive oxidation of proteins such as lysozyme and ferritin does not increase their ubiquitinylation, yet the oxidized forms of both proteins are preferentially degraded by proteasome. Furthermore, rates of oxidized protein degradation by cell lysates are not significantly altered by addition of ATP, excluding the possibility of an energy requirement for this pathway. Contrary to earlier popular belief that most proteasomal degradation is conducted by the 26 S proteasome with ubiquitinylated substrates, our work suggests that oxidized proteins are degraded without ubiquitin conjugation (or ATP hydrolysis) possibly by the 20 S proteasome, or the immunoproteasome, or both.
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PMID:Ubiquitin conjugation is not required for the degradation of oxidized proteins by proteasome. 1240 7

The ubiquitin-proteasome pathway is critically involved in the pathology of neurodegenerative diseases characterized by protein misfolding and aggregation. Data in the present study suggest that the polyglutamine neurodegenerative disease protein, ataxin-3 (AT3), functions in the ubiquitin-proteasome pathway. AT3 contains an ubiquitin interaction motif (UIM) domain that binds polyubiquitylated proteins with a strong preference for chains containing four or more ubiquitins. Mutating the conserved leucine in the first UIM (L229A) almost totally eliminates binding to polyubiquitin chains while a similar mutation in the second UIM (L249A) also inhibits binding to polyubiquitin chains but to a lesser extent. Both wild-type and pathological AT3 increase cellular levels of a short-lived GFP that is degraded by the ubiquitin-proteasome pathway. AT3 has several properties characteristic of ubiquitin proteases including decreasing polyubiquitylation of 125I-lysozyme by removing ubiquitin from polyubiquitin chains, cleaving a ubiquitin protease substrate, and binding the specific ubiquitin protease inhibitor, ubiquitin-aldehyde. Mutating the predicted catalytic cysteine in AT3 inhibits each of these ubiquitin protease activities. The ability to bind and cleave ubiquitylated proteins is consistent with AT3 playing a role in the ubiquitin-proteasome system. This raises the possibility that pathological AT3, which tends to misfold and aggregate, may be exposed to aggregate-prone misfolded/denatured proteins as part of its normal function.
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PMID:The polyglutamine neurodegenerative protein ataxin-3 binds polyubiquitylated proteins and has ubiquitin protease activity. 1455 76

Recently, impairment of the ubiquitin-proteasome system is suggested to be responsible for the neuronal death in ageing and Parkinson's disease. The specific degeneration of dopamine neurons containing neuromelanin (NM) suggests that NM itself may be involved in the cellular dysfunction and death, even though the direct link has never been reported. We examined the effects of NM isolated from the human substantia nigra on the proteasome activity in human dopaminergic SH-SY5Y cells. NM reduced the activities of 26S proteasome, as shown in situ using a green fluorescent protein homologue targeted to 26S proteasome and also in vitro using ubiquitinated lysozyme as a substrate. However, NM did not affect 20S proteasome activity in vitro. NM reduced the amount of PA700 regulatory subunit of 26S proteasome, but did not affect that of alpha- and beta-subunits of 20S proteasome. These results suggest that NM may inhibit the ubiquitin-26S proteasome system, and determine the selective vulnerability of dopamine neurons in ageing and related disorders.
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PMID:Neuromelanin inhibits enzymatic activity of 26S proteasome in human dopaminergic SH-SY5Y cells. 1548 Aug 37

Cell-based vaccines consisting of invariant chain-negative tumor cells transfected with syngeneic MHC class II (MHC II) and costimulatory molecule genes are prophylactic and therapeutic agents for the treatment of murine primary and metastatic cancers. Vaccine efficacy is due to direct presentation of endogenously synthesized, MHC II-restricted tumor peptides to CD4+ T cells. Because the vaccine cells lack invariant chain, we have hypothesized that, unlike professional APC, the peptide-binding groove of newly synthesized MHC II molecules may be accessible to peptides, allowing newly synthesized MHC II molecules to bind peptides that have been generated in the proteasome and transported into the endoplasmic reticulum via the TAP complex. To test this hypothesis, we have compared the Ag presentation activity of multiple clones of TAP-negative and TAP-positive tumor cells transfected with I-Ak genes and the model Ag hen egg white lysozyme targeted to the endoplasmic reticulum or cytoplasm. Absence of TAP does not diminish Ag presentation of three hen egg white lysozyme epitopes. Likewise, cells treated with proteasomal and autophagy inhibitors are as effective APC as untreated cells. In contrast, drugs that block endosome function significantly inhibit Ag presentation. Coculture experiments demonstrate that the vaccine cells do not release endogenously synthesized molecules that are subsequently endocytosed and processed in endosomal compartments. Collectively, these data indicate that vaccine cell presentation of MHC II-restricted endogenously synthesized epitopes occurs via a mechanism independent of the proteasome and TAP complex, and uses a pathway that overlaps with the classical endosomal pathway for presentation of exogenously synthesized molecules.
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PMID:Presentation of endogenously synthesized MHC class II-restricted epitopes by MHC class II cancer vaccines is independent of transporter associated with Ag processing and the proteasome. 1569 7

To study the interaction between an invading virus and its host, we investigated differential gene expression in mandarin fish Siniperca chuatsi experimentally infected with infectious spleen and kidney necrosis virus (ISKNV). Subtractive cDNA libraries were constructed by suppression subtractive hybridization (SSH) from spleens of mock- and ISKNV-infected fish. Both forward- and reverse-subtracted libraries were generated. In the forward library, genes of the ubiquitin-proteasome proteolytic pathway, defense-related genes, a cytoskeletal protein gene, an apoptosis-related gene encoding inhibitor of apoptosis protein and JFC/EBPb cDNA for CAAT/Enhancer binding protein beta were up-regulated after infection. In the reverse library, genes that encoded CD59/neurotoxin/Ly-6-like protein, carboxypeptidase A2, and goose-type lysozyme were down-regulated. Some of these genes were analyzed by reverse transcription-polymerase chain reaction to confirm their differential expression as a result of virus infection. The results of this study may contribute to our understanding of fish innate immune response to ISKNV.
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PMID:Differential gene expression profile in spleen of mandarin fish Siniperca chuatsi infected with ISKNV, derived from suppression subtractive hybridization. 1726 Aug 30


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