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 proteasome is responsible for the non-lysosomal degradation of misfolded, transient, or ubiquitin-tagged proteins. This fact and the identification of two major-histocompatibility-complex-(MHC)-encoded proteasomal subunits, LMP2/7, suggest an important role of the proteasome in antigen processing. Using purified 20S proteasomes from a wild-type and a LMP2/7-deletion T lymphoblastoid cell line, we analyzed the effect of LMP2/7 on the peptidase and proteolytic activities of the complex in the context of various purification and activation methods. The incorporation of LMP2/7 alters the peptidase activity against fluorogenic substrates, but these effects are not reflected in the time-dependent degradation pattern of oxidized insulin B chain or of peptide epitopes of an antigenic protein. No effect of LMP2/7 on the degradation pattern of these substrates was observed by either reverse-phase chromatography, pool sequencing, or mass spectrometry. The 20S proteasome can cleave insulin B chain at nearly every position, showing that the P1 position alone does not determine the cleavage sites. The maximum of the length distribution of the end products, makes these ideal candidates for MHC display; yet we find that a natural epitope derived from human histone H3 is further degraded by 20S proteasomes. Alanine scans and substitutions with related amino acids of this epitope indicate that, as in insulin B chain, the cleavage sites are not determined by the P1 position alone.
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PMID:Effects of major-histocompatibility-complex-encoded subunits on the peptidase and proteolytic activities of human 20S proteasomes. Cleavage of proteins and antigenic peptides. 863 60

Activation of transcription factor NF-kappaB involves signal-induced degradation of the protein inhibitor IkappaB-alpha and release of NF-kappaB which translocates to the nucleus where it influences transcription of responsive genes. Although multiple regions of IkappaB-alpha are involved in this process, the N-terminal region of the protein has been identified as a regulatory region that is required for signal induced phosphorylation and degradation. The sensitivity of IkappaB-alpha degradation to peptide aldehydes which inhibit components of the proteasome and the detection of ubiquitinated forms of IkappaB-alpha indicate that IkappaB-alpha is degraded by the ubiquitin-proteasome pathway. To identify lysine residues that represent the sites of ubiquitin addition, a series of lysine to arginine mutations were introduced into IkappaB-alpha and the mutant proteins tested for their ability to function in vivo. Exposure of COS7 cells, cotransfected with IkappaB-alpha and a TNF-responsive NF-kappaB reporter gene, resulted in stimulation of reporter activity as a consequence of IkappaB-alpha degradation. In contrast, this effect was drastically reduced when an IkappaKB-alpha mutant carrying serine to alanine changes at amino-acids, 32 and 36, which blocks both signal-induced phosphorylation and ubiquitin conjugation of the protein, was co-transfected with the reporter gene. Likewise, a mutant form of IkappaB-alpha containing lysine to arginine changes at positions 21 and 22 (K21R, K22R) severely reduces TNF-induced activation of the NF-kappaB-dependent reporter gene. Examination of the metabolism of mutant IkappaB-alpha molecules reveals that, while the K21R, K22R mutant inhibits the DNA-binding activity of NF-kappaB and undergoes signal induced phosphorylation, it is neither ubiquitinated nor degraded in response to TNF. Thus, it is likely that after signal-induced phosphorylation Of IkappaB-alpha on serine residues 32 and 36, lysine residues 21 and 22 are major sites of ubiquitin ligation which target the protein for rapid degradation by the proteasome.
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PMID:Identification of lysine residues required for signal-induced ubiquitination and degradation of I kappa B-alpha in vivo. 864 84

NF-kappa B is a dimeric protein that serves to initiate gene transcription in higher eukaryotic cells in response to mainly pathogenic stimuli. Its activity is controlled by a third inhibitory subunit, called I kappa B. When I kappa B is bound, NF-kappa B cannot bind to DNA or enter the nucleus but is stored in a latent cytoplasmic form. Upon stimulation of cells I kappa B is released, which allows the activation of NF-kappa B. We have analyzed the molecular mechanism underlying the removal of I kappa B-alpha. Distinct extracellular stimuli lead to a phosphorylation of I kappa B-alpha of serines 32 and 36 by a yet unidentified kinase. These modifications do not directly dissociate I kappa B from NF-kappa B but render the inhibitor highly susceptible for proteolytic degradation by, presumably, the proteasome. In this paper, we report for the first time that higher molecular mass forms of I kappa B-alpha occur under conditions that lead to a phosphorylation of I kappa B-alpha and activation of NF-kappa B. These I kappa B-alpha variants had discrete molecular masses and were most prominent in cells overexpressing I kappa B-alpha, suggesting the covalent modification of I kappa B-alpha by ubiquitin conjugation. The proteasome inhibitor Cbz-Ile-Glu(O-t-Bu)-Ala-leucinal (PSI), which stabilizes the phospho form of I kappa B-alpha, only slightly increased the amount of conjugates indicating that the conjugation of I kappa B-alpha with ubiquitin was the rate-limiting step in I kappa B-alpha degradation, and not its phosphorylation or proteolysis. Our data suggest that conjugation of I kappa B-alpha with ubiquitin is an intermediate reaction in the phosphorylation-controlled degradation of I kappa B-alpha and the subsequent activation of NF-kappa B.
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PMID:Appearance of apparently ubiquitin-conjugated I kappa B-alpha during its phosphorylation-induced degradation in intact cells. 865 51

We have studied the degradation of a set of long peptides (9-30 amino acids) from the nucleoprotein of influenza A. In common for all these peptides is the core sequence NH2-Ser-Arg-Tyr-Trp-Ala-Ile-Arg-Thr-Arg-COOH, NP383-391, known as an antigenic peptide specific for the HLA-B27 class I antigen. We show that this peptide is generated by enriched cytosolic proteasomes of two sizes, 20S and 12S. The 12S proteasome is the precursor, the preproteasome, to the 20S mature proteasome as shown by pulse-chase experiment and is most likely responsible for the proteolytic activity in the 12S region. Cleavage at the N-terminus is distinct and restricted to residue 383, independent of the N-terminal extension of the peptide. The C-terminus is generated via cleavage at three sites. Intermediate and final peptide products were identified by mass spectrometry. Finally, we show that the NP383-391 peptide generated by proteasomes in vitro is functional inasmuch as it possesses the ability to stimulate assembly of in vitro translated HLA-B27 antigens.
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PMID:Proteasomes generate in vitro a natural peptide of influenza-A nucleoprotein functional in HLA-B27 antigen assembly. 867 33

Bryostatins and phorbol esters acutely activate and subsequently down-regulate protein kinase C (PKC) by inducing its proteolysis via an unknown pathway. Here we show that treatment of renal epithelial cells with bryostatin 1 (Bryo) produced novel PKC-alpha species, which were larger than the native protein (80 kDa). The >80 kDa PKC-alpha species contained Ubi as indicated by immunostaining and accumulated in the presence of lactacystin, a selective inhibitor of proteolysis by the proteasome. In vitro experiments with 125I-ubiquitin and membranes from Bryo-treated cells showed that PKC-alpha became ubiquitinated by a reaction that depended on ATP and a cytosolic fraction. Lactacystin or a peptidyl aldehyde, Bz-Gly-Leu-Ala-leucinal, which inhibits certain proteinase activities of the proteasome, inhibited Bryo-evoked disappearance of PKC-alpha protein from the cells. Lacta preserved Bryo-induced 32P-labeled PKC-alpha indicating that the proteasome inhibitor spared activated enzyme from down-regulation in vivo. These findings show that Bryo induces the degradation of PKC-alpha by the ubiquitin-proteasome complex.
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PMID:Ubiquitination of protein kinase C-alpha and degradation by the proteasome. 870 57

HeLa cells were treated with different concentrations of an inhibitor of the proteasome chymotrypsin-like activity, the peptidyl aldehyde N-benzyloxycarbonyl-Ile-Glu(O-t-butyl)-Ala-leucinal (PSI). A detailed analysis, which included flow cytometry, cell counting and morphological assessment, was performed. PSI treatment induces a significant reduction of mitotic activity, accompanied by metaphase arrest of the mitotic cells. DNA flow cytometry shows an accumulation of the cells in G2+M phases of the cell cycle, which indicates the existence of a proteasome-mediated step in the G2-phase of the cell cycle. After removal of the inhibitor and supplementation with fresh medium, the cell cycle is resumed, but the mitotic cells show increased misalignment of chromosomes in the metaphase plate. PSI also induces HeLa cells to acquire a fibroblastoid phenotype.
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PMID:An inhibitor of the chymotrypsin-like activity of the multicatalytic proteinase complex (20S proteasome) induces arrest in G2-phase and metaphase in HeLa cells. 879 90

Recent work suggests that the proteolytic degradation of the nuclear lamins is a common event in apoptosis, although the nature of the proteases involved is still not clear. Our previous work showed that the degradation of lamin B1 in glucocorticoid-treated thymocytes occurs via a Ca2+-sensitive mechanism and that exogenous Ca2+ promotes lamin degradation in isolated thymocyte nuclei from untreated cells. Here we demonstrate that peptide-based inhibitors of the interleukin 1beta-converting enzyme family of cysteine proteases (Tyr-Val-Ala-Asp fluoromethyl ketone) and of the nuclear scaffold multicatalytic proteinase (Ala-Pro-Phe chloromethyl ketone) block the degradation of lamin B1 to a 21-kDa fragment in thymocytes treated with glucocorticoid, the Ca2+-mobilizing agent thapsigargin, or antibodies to the T cell receptor. However, among a panel of inhibitors specific for several different proteases implicated in apoptosis, only tosylphenylalanyl chloromethyl ketone and the nuclear scaffold protease inhibitor block lamin degradation, histone H1 cleavage, and DNA fragmentation in isolated thymocyte nuclei incubated with Ca2+. Overexpression of human BCL-2 in nuclei by stable transfection resulted in an inhibition of Ca2+-stimulated lamin degradation and DNA fragmentation, suggesting that endogenous nuclear BCL-2 regulates activation of the nuclear scaffold protease. The results demonstrate the existence of an alternative pathway of lamin degradation and DNA fragmentation mediated by a resident Ca2+-stimulated nuclear protease that is not directly dependent upon activation of the interleukin 1beta-converting enzyme family of cell death regulators.
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PMID:Calcium-dependent, interleukin 1-converting enzyme inhibitor-insensitive degradation of lamin B1 and DNA fragmentation in isolated thymocyte nuclei. 879 2

The aim of the present study was to characterize human CYP2E1 turnover and examine the possible role of the proteasome proteolytic pathway in the rapid degradation of CYP2E1 in a transfected HepG2 cell line expressing human CYP2E1. Microsomes isolated from MVh2E1-9 cells catalyzed a slow degradation of the expressed CYP2E1, which was prevented by the addition of 4-methylpyrazole, a ligand which stabilizes CYP2E1. The addition of the cytosolic fraction of the HepG2 cells to the microsomes produced rapid degradation of CYP2E1. This rapid degradation required MgATP and was completely prevented by 4-methylpyrazole. Pulse-chase experiments after labeling CYP2E1 with [35S]-methionine and immunoprecipitation with anti-human CYP2E1 IgG indicated a biphasic turnover of CYP2E1 with half-lives of 2.5 and 6 hours. The addition of Czb-Ile-Glu(OtBu)-Ala-Leucinal(PSI) as a cell penetrating proteasome inhibitor, at concentrations ranging from 5 to 80 microM resulted in protection against the degradation of CYP2E1. PSI also increased the steady state accumulation of CYP2E1, consistent with its inhibition of CYP2E1 turnover. These results suggest that the proteasome complex plays a major role in the degradation of human CYP2E1 in the transfected HepG2 cells.
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PMID:Role of the proteasome complex in degradation of human CYP2E1 in transfected HepG2 cells. 883 79

A yeast screen was developed to identify mutations in human cyclin E that lead to stabilization of the protein in order to identify determinants important for cyclin E turnover. Both C-terminal truncations and missense mutations near the C-terminus of cyclin E conferred hyperstability in vivo, suggesting that sequences in this region were critical for turnover. The following observations indicate that autophosphorylation of CDK2/cyclin E on Thr380 of the cyclin regulates cyclin E destruction: (i) mutation of Thr380 to Ala stabilizes cyclin E in yeast and mammalian cells; (ii) cyclin E/CDK2 autophosphorylates on cyclin E in vitro and cyclin E is a phosphoprotein in vivo in mammalian cells; (iii) the T380A mutation eliminates phosphorylation on the same site in mammalian cells and in vitro; (iv) inhibiting CDK2 activity in vivo stabilizes cyclin E; (v) the T380A mutation prevents ubiquitination of cyclin E. These results suggest a model where activation of cyclinE/CDK2 is coupled to cyclin E turnover via site-specific phosphorylation, which acts as a signal for ubiquitination and proteasome processing.
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PMID:Activation of cyclin E/CDK2 is coupled to site-specific autophosphorylation and ubiquitin-dependent degradation of cyclin E. 886 47

Corn peptide (CP) was prepared from corn gluten meal by proteolysis with alkaline protease from alkalophilic Bacillus A-7. Free amino acids were not found in the CP product. Gel filtration on a Shodex OH-packed column revealed that the molecular weight distribution of the CP was less than about 2,000, characteristic of dipeptides to decapeptides, i.e. oligopeptides. The amino acid pattern of CP was similar to that of corn gluten meal, which was rich in alanine and branched-chain amino acids, but poor in basic amino acids. The effect of the CP administration on alcohol metabolism was examined with SHR-SP, which were given ethanol orally through a gastric tube at the rate of 1.0 g/kg. Prior administration of CP at 1.0 g/kg resulted in fast disappearance of ethanol and its oxidative product acetaldehyde from the blood relative to the control without administration. Hence, it is suggested that CP, rather than its constituent amino acids such as alanine and proline, effectively takes part in enhancing the metabolism of ethanol as well as acetaldehyde.
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PMID:Preparation of corn peptide from corn gluten meal and its administration effect on alcohol metabolism in stroke-prone spontaneously hypertensive rats. 886 58


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