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At least 19 recordsLinked to original sources
PubMed2007

Mechanism for the degradation of origin recognition complex containing Orc5p with a defective Walker A motif and its suppression by over-production of Orc4p in yeast cells.

Orc5p is one of six subunits constituting the ORC (origin recognition complex), a possible initiator of chromosomal DNA replication in eukaryotes. Orc5p contains a Walker A motif. We recently reported that a strain of Saccharomyces cerevisiae having a mutation in Orc5p's Walker A motif (orc5-A), showed cell-cycle arrest at G2/M and degradation of ORC at high temperatures (37 degrees C). Over-production of Orc4p, another subunit of ORC, specifically suppressed these phenotypes [Takahashi, Yamaguchi, Yamairi, Makise, Takenaka, Tsuchiya and Mizushima (2004) J. Biol. Chem. 279, 8469-8477]. In the present study, we examined the mechanisms of ORC degradation and of its suppression by Orc4p over-production. In orc5-A, at high temperatures, ORC is degraded by proteasomes; either addition of a proteasome inhibitor, or introduction of a mutation of either tan1-1 or nob1-4 that inhibits proteasomes, prevented ORC degradation. Introduction of the tan1-1 mutation restored cell cycle progression, suggesting that the defect was due to ORC degradation by proteasomes. Yeast two-hybrid and co-immunoprecipitation analyses suggested that Orc5p interacts preferentially with Orc4p and that the orc5-A mutation diminishes this interaction. We suggest that this interaction is mediated by the C-terminal region of Orc4p, and the N-terminal region of Orc5p. Based on these observations, we consider that ATP binding to Orc5p is required for efficient interaction with Orc4p and that, in orc5-A, loss of this interaction at higher temperatures allows proteasomes to degrade ORC, causing growth defects. This model could also explain why over-production of Orc4p suppresses the orc5-A strain's phenotype.

Adenosine Triphosphate↗
PubMed1993

Reaction of proteasomes with peptidylchloromethanes and peptidyldiazomethanes.

The multicatalytic endopeptidase complex (proteasome) has multiple distinct peptidase activities. These activities have often been referred to as 'chymotrypsin-like', 'trypsin-like' and 'peptidylglutamyl-peptide hydrolase' activities according to the type of residue in the P1 position, although it is now clear that mammalian proteasomes have at least five distinct catalytic sites. In the present study, potential affinity-labelling reagents (peptidylchloromethanes, peptidyldiazomethanes, a peptidylfluoromethane and peptidylsulphonium salts) containing hydrophobic, basic or acidic amino acid residues in the P1 position have been tested for inhibition of the different activities of the rat liver proteinase complex. The results show that individual peptidase activities of proteasomes can be inhibited by a variety of peptidylchloromethanes and peptidyldiazomethanes. Although the rate of inactivation of proteasomes by even the most effective peptidylchloromethanes and peptidyldiazomethanes are often quite slow (k(obs)/[I] in the range 0.1-10 M-1 x s-1) compared with the reaction of similar compounds with some other proteinases, the results provide useful information concerning the specificity of the distinct catalytic centres of proteasomes, and some selective affinity-labelling reagents have been identified. Tyr-Gly-Arg-chloromethane was found to be a useful inhibitor of trypsin-like activity. Inhibition of the other peptidase activities was often incomplete, even after repeated addition of inhibitor, and it proved to be difficult to predict the effect of different reagents. For example, Cbz-Tyr-Ala-Glu-chloromethane was found to inhibit 'chymotrypsin-like' activity (assayed with Ala-Ala-Phe-7-amino-4-methylcoumarin or succinyl-Leu-Leu-Val-Tyr-7-amino-4-methylcoumarin), while the best inhibitors of 'peptidylglutamyl-peptide hydrolase' activities (assayed with benzyloxycarbonyl-Leu-Leu-Glu beta-naphthylamide) were peptidyldiazomethanes containing hydrophobic amino acid residues. These results suggest that the original nomenclature of proteasome activities is misleading, because the residue in the P1 position is not the only determinant of specificity.

Affinity Labels↗
PubMedDate not supplied

Proteasome workshop at Colorado State University. Fort Collins, Colo., July 1-3, 1995. Report and abstracts.

An international workshop on the proteasome was held on the campus of Colorado State University, Fort Collins, July 1-3, 1995. The program consisted of five oral sessions and one poster session. The abstracts of the posters and many of the oral presentations appear in this issue of Enzyme and Protein. The oral sessions were organized around four themes: structure and catalytic properties; allosteric regulators and synthetic inhibitors; the 26S proteasome; and physiological functions, developmental expression, and endogenous substrates. Since entire issues of Enzyme and Protein [vol. 47, No. 4-6, 1993] and Molecular Biology Reports [vol. 21, No. 1, 1995] have been devoted to the proteasome, this summary emphasizes recent results and current research of many of the laboratories studying this fascinating complex.

Cysteine Endopeptidases↗
PubMed1998

Constitutive and interferon-gamma-induced expression of the human proteasome subunit multicatalytic endopeptidase complex-like 1.

Proteasomes generate peptides from intracellular endogenous and viral proteins for presentation by MHC class I molecules. During viral infection, interferon-gamma (IFN-gamma) acts as a cytokine altering the catalytic specificity of proteasomes by inducing the synthesis of the three proteasome subunits, low molecular weight protein (LMP) 2, LMP7 and multicatalytic endopeptidase complex-like 1 (MECL1). LMP2 and LMP7 have been shown to favour the presentation of certain antigenic peptides. These subunits are constitutively expressed in cell lines related to the immune system and IFN-gamma-inducible in other cell lines. Less is known about MECL1. To reveal the extent of constitutive and IFN-gamma-induced expression of MECL1, we studied MECL1 in different cell lines by Northern and Western blotting. The two B cell lines IM9 and Reh showed high constitutive expression of MECL1, only slightly induced by IFN-gamma stimulation. The B cell line Daudi and the monocyte cell line THP-1 expressed MECL1 constitutively at an intermediate level. The MECL1 protein level in the THP-1 cells increased markedly in response to IFN-gamma. In cells unrelated to the immune system, a very low constitutive expression of MECL1 was detected, highly inducible by IFN-gamma. These results indicate that, similar to LMP2 and LMP7, MECL1 is constitutively expressed at high levels only in certain cell lines and can be induced by IFN-gamma in other cell lines. The differential expression of MECL1 may be of importance for which antigenic peptides are presented by different cells as well as by the same cells at different IFN-gamma levels.

Adenocarcinoma↗
PubMed1999

Proteasomes and other self-compartmentalizing proteases in prokaryotes.

The proteasome represents the major non-lysosomal proteolytic system in eukaryotes. It confines proteolytic activity to an inner compartment that is accessible to unfolded proteins only. The strategy of controlling intracellular breakdown of proteins by self-compartmentalization is also used by different types of prokaryotic energy-dependent proteases. Genomic sequencing data reveal that various combinations of these energy-dependent proteases occur in prokaryotic cells from different lineages.

Adenosine Triphosphatases↗
PubMed1997

Ubiquitination of Na,K-ATPase alpha1 and alpha2 subunits.

Na,K-ATPase, an essential transporter of mammalian cells, is an oligomeric transmembrane protein composed of two subunits, alpha and beta, of which there are several isoforms. In this study, we demonstrate that the alpha1 and alpha2 isoforms of the Na,K-ATPase alpha subunit are modified by the covalent attachment of ubiquitin polymers in COS-7 cells. We propose that polyubiquitination of the Na,K-ATPase alpha subunit may play a role in regulating its degradation.

Animals↗
PubMedDate not supplied

Production of alkaline proteinase by a new bacterial strain, Bacillus PE-11.

A new strain of Bacillus sp., Bacillus PE-11 isolated and identified in our laboratory was found to be a potential producer of alkaline proteinase. The production of proteinase was studied in thirteen different reported media to select the optimal production medium. In all the reported media, the medium XII of composition: glucose, 0.2%; peptone, 1.5%; salt solution, 5% (MgSO4. 7H2O, 0.5%; KH2PO4, 0.5%; FeSO4. 7H2O, 0.01%; distilled water make up to 100 ml.) is found to have the highest inducing effect on enzyme production.

Bacillus↗
PubMed1999

The 26S proteasome: a molecular machine designed for controlled proteolysis.

In eukaryotic cells, most proteins in the cytosol and nucleus are degraded via the ubiquitin-proteasome pathway. The 26S proteasome is a 2.5-MDa molecular machine built from approximately 31 different subunits, which catalyzes protein degradation. It contains a barrel-shaped proteolytic core complex (the 20S proteasome), capped at one or both ends by 19S regulatory complexes, which recognize ubiquitinated proteins. The regulatory complexes are also implicated in unfolding and translocation of ubiquitinated targets into the interior of the 20S complex, where they are degraded to oligopeptides. Structure, assembly and enzymatic mechanism of the 20S complex have been elucidated, but the functional organization of the 19S complex is less well understood. Most subunits of the 19S complex have been identified, however, specific functions have been assigned to only a few. A low-resolution structure of the 26S proteasome has been obtained by electron microscopy, but the precise arrangement of subunits in the 19S complex is unclear.

Cysteine Endopeptidases↗
PubMedDate not supplied

Assays of proteasome activity in relation to aging.

Proteasomes play a major role in intracellular protein turnover. They exist in cells in several different molecular forms including 20S proteasomes, 26S proteasomes and PA28-20S proteasome complexes. In this study we have compared the properties of these purified proteasome complexes to try to design assays that will distinguish between the different complexes (26S proteasome, 20S proteasome, PA28-20S proteasome) in cell extracts. Although the different purified complexes were found to have differences in stability, and in their sensitivity to low concentrations of SDS and salt, the results suggest that it is not straightforward to assay selectively for each type of complex in cell extracts. The relative contribution of different proteasome complexes varies in different cell types and there may be other proteases present which hydrolyse the chosen substrate. Proteasome assays carried out under defined conditions allow comparisons of activity in cell extracts as a function of age, but separation by gel filtration on a Superose 6 column was found to be a useful method for determining the level of different proteasome related complexes.

Aging↗