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)

As an attempt to recruit the third calcium binding site of thermitase into subtilisin BL, a Bacillus lentus alkaline protease (BLAP), the amino acid sequence from position 50 to 60 and position 92 was modified to the equivalent amino acids in thermitase. The resulting protein, designated BLAPm109, exhibited unusual biochemical features. Peptide mapping and gel electrophoresis revealed that two protein species co-purify in a ratio of about 1:1. Form 1 consisted of a single polypeptide of 269 amino acid residues. Form 2 was the same protein but with an internal peptide bond cleavage at the C-terminus of position 54. On electropherograms a dimer of Form 1 and Form 2 was also detectable. A zymogram showed that all three molecular species were catalytically active. From this protein mixture, crystals suitable for X-ray analysis were nevertheless obtained. SDS-PAGE of protein recovered from a crystal revealed that only Form 2 appears. in the crystal. The space group for this crystal was P21 with unit cell dimensions of a=42 angstroms, b=58 angstroms, c=47 angstroms and beta = 106.3 degrees. Examination of the preliminary electron density map revealed that the "thermitase loop" from 50 to 60 departs from the surface of the protein and winds through the active site of a symmetry-related copy of the asymmetric unit.
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PMID:Unusual ligand binding at the active site domain of an engineered mutant of subtilisin BL. 879 30

Degradation of proteins that are retained in the quality control apparatus of the endoplasmic reticulum (ER) has been attributed to a third proteolytic system, distinct from the lysosomal and the cytoplasmic ubiquitin-dependent proteosomal proteolytic pathways. However, several recent studies have shown that ER degradation of a mutant membrane protein, CFTRdeltaF508, is at least in part mediated from the cytoplasmic side by the 26 S proteasome. In this study, we examined the possibility that ER degradation of mutant secretory protein alpha1-antitrypsin (alpha1-AT) Z, the mutant protein associated with infantile liver disease and adult-onset emphysema of alpha1-AT deficiency, is mediated by the proteasome. The results show that a specific proteasome inhibitor, lactacystin, inhibits ER degradation of alpha1-ATZ in transfected human fibroblast cell lines and in a cell-free microsomal translocation system. Although it is relatively easy to conceptualize how a transmembrane protein like CFTRDeltaF508 might be accessible on the cytoplasmic aspect of the ER membrane for ubiquitination and degradation by the proteasome, it is more difficult to conceptualize how this might occur for a luminal polypeptide. The results show that, once within the lumen of the ER, alpha1-ATZ interacts with the transmembrane molecular chaperone calnexin and specifically induces the polyubiquitination of calnexin. The results, therefore, provide evidence that the proteasome, from its cytoplasmic localization, induces the degradation of the luminal alpha1-ATZ molecule by first attacking the cytoplasmic tail of calnexin molecules that are associated with alpha1-ATZ.
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PMID:Degradation of a mutant secretory protein, alpha1-antitrypsin Z, in the endoplasmic reticulum requires proteasome activity. 879 55

Proteasomes are the multi-subunit protease thought to play a key role in the generation of peptides presented by major histocompatibility complex (MHC) class I molecules. When cells are stimulated with interferon gamma, two MHC-encoded subunits, low molecular mass polypeptide (LMP) 2 and LMP7, and the MECL1 subunit encoded outside the MHC are incorporated into the proteasomal complex, presumably by displacing the housekeeping subunits designated Y, X, and Z, respectively. These changes in the subunit composition appear to facilitate class I-mediated antigen presentation, presumably by altering the cleavage specificities of the proteasome. Here we show that the mouse gene encoding the Z subunit (Psmb7) maps to the paracentromeric region of chromosome 2. Inspection of the mouse loci adjacent to the Psmb7 locus provides evidence that the paracentromeric region of chromosome 2 and the MHC region on chromosome 17 most likely arose as a result of a duplication that took place at an early stage of vertebrate evolution. The traces of this duplication are also evident in the homologous human chromosome regions (6p21.3 and 9q33-q34). These observations have implications in understanding the genomic organization of the present-day MHC and offer insights into the origin of the MHC.
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PMID:Chromosomal localization of the proteasome Z subunit gene reveals an ancient chromosomal duplication involving the major histocompatibility complex. 879 60

The ocular lens consists of a single layer of epithelial cells on its anterior surface and underlying fiber cells, which are derived from the epithelial cells by differentiation and make up the bulk of the lens. Because lens cells are segregated by age and stage of differentiation, we are using this tissue to study the role of the proteasome in differentiation. The purpose of this study is to corroborate the ATPase function of chick subunit 4 (cS4) and assess the levels of the mRNA in the differentiating lens relative to other tissues. We have generated a computer model of the tertiary structure of the ATPase domain of the cS4 of the ATPase complex that regulates the 20S proteasome. The predicted polypeptide from the cloned cDNA of cS4 (440 residues) had a calculated molecular mass of 49,182 and is 98 and 73% identical to human and yeast S4 protein sequences, respectively. A computer search for comparison with known proteins in GenBank showed that the cS4 protein sequence has a conserved region of about 200 amino acid residues including an ATP/GTP binding site and a mitochondrial energy transfer proteins signature sequence. Based on secondary structure, the computer-generated model of the ATPase domain is comparable to that of RecA, with a root mean square deviation of 0.851 from the RecA triad. mRNA in the 14-day-old chick embryo lens is derived primarily (90%) from differentiating cells. The level of cS4 mRNA determined by quantitative RT/PCR in this differentiating tissue was comparable to the cS4 mRNA levels in chick liver, heart, and brain.
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PMID:cDNA cloning of a chick homologue of human ATPase complex subunit 4, quantitative tissue distribution and tertiary structure comparison of the ATPase domain to RecA. 880 79

The 26S proteasome is a large multisubunit protease complex, the largest regulatory subunit of which is a component named p112. Molecular cloning of cDNA encoding human p112 revealed a polypeptide predicted to have 953 amino acid residues and a molecular mass of 105,865. The human p112 gene was mapped to the q37.1-q37.2 region of chromosome 2. Computer analysis showed that p112 has strong similarity to the Saccharomyces cerevisiae Sen3p, which has been listed in a gene bank as a factor affecting tRNA splicing endonuclease. The SEN3 also was identified in a synthetic lethal screen with the nin1-1 mutant, a temperature-sensitive mutant of NIN1. NIN1 encodes p31, another regulatory subunit of the 26S proteasome, which is necessary for activation of Cdc28p kinase. Disruption of the SEN3 did not affect cell viability, but led to temperature-sensitive growth. The human p112 cDNA suppressed the growth defect at high temperature in a SEN3 disruptant, indicating that p112 is a functional homologue of the yeast Sen3p. Maintenance of SEN3 disruptant cells at the restrictive temperature resulted in a variety of cellular dysfunctions, including defects in proteolysis mediated by the ubiquitin pathway, in the N-end rule system, in the stress response upon cadmium exposure, and in nuclear protein transportation. The functional abnormality induced by SEN3 disruption differs considerably from various phenotypes shown by the nin1-1 mutation, suggesting that these two regulatory subunits of the 26S proteasome play distinct roles in the various processes mediated by the 26S proteasome.
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PMID:CDNA cloning of p112, the largest regulatory subunit of the human 26s proteasome, and functional analysis of its yeast homologue, sen3p. 881 93

The UBC9 gene of the yeast Saccharomyces cerevisiae is essential for cell viability and encodes a soluble protein of the nucleus that is metabolically stable. Products of mutant alleles selected to confer temperature-sensitive in vivo function were found to be extremely short-lived at the restrictive but long-lived at the permissive condition. An extragenic suppressor mutation was isolated which increased thermoresistance of a ubc9-1 strain. This suppressor turned out to stabilize the mutated gene product, indicating that the physiological activity of ubc9-1 protein is primarily controlled by conditional proteolysis. The labile ubc9-1 protein appears to be a substrate for ubiquitination, and its turnover was substantially reduced by expression of a ubiquitin derivative that interferes with formation of multi-ubiquitin chains. Stabilization resulted also from competitive inhibition of Ubc4-related ubiquitin-conjugating enzymes. Activity of the proteasome complex was crucial to rapid breakdown, whereas vacuolar proteases were dispensable. Thus, the heat-denatured ubc9-1 protein is targeted for proteolysis by the ubiquitin-proteasome pathway and may serve as a useful tool to further define the process by which a misfolded polypeptide is recognized.
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PMID:A yeast Ubc9 mutant protein with temperature-sensitive in vivo function is subject to conditional proteolysis by a ubiquitin- and proteasome-dependent pathway. 882 7

A growing number of cellular regulatory mechanisms are being linked to protein modification by the polypeptide ubiquitin. These include key transitions in the cell cycle, class I antigen processing, signal transduction pathways, and receptor-mediated endocytosis. In most, but not all, of these examples, ubiquitination of a protein leads to its degradation by the 26S proteasome. Following attachment of ubiquitin to a substrate and binding of the ubiquitinated protein to the proteasome, the bound substrate must be unfolded (and eventually deubiquitinated) and translocated through a narrow set of channels that leads to the proteasome interior, where the polypeptide is cleaved into short peptides. Protein ubiquitination and deubiquitination are both mediated by large enzyme families, and the proteasome itself comprises a family of related but functionally distinct particles. This diversity underlies both the high substrate specificity of the ubiquitin system and the variety of regulatory mechanisms that it serves.
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PMID:Ubiquitin-dependent protein degradation. 898 60

Although the structure of the 20 S proteasome from Thermoplasma acidophilum has been elucidated, its enzymatic properties have not been explored in depth. Thermoplasma proteasomes, which contain one type of active site, exhibit not only "chymotrypsin-like" activity (as reported), but also some "post-glutamyl" and "trypsin-like" activities. Like eukaryotic proteasomes, its activity can be stimulated by SDS, Mg2+, and also guanidine HCl, but not urea. The enzyme was strongly inhibited by novel peptide aldehydes with hydrophobic P4 residues, and was rapidly inactivated by 3, 4-dichloroisocoumarin (DCI). DCI modified the N-terminal threonine of the catalytic beta-subunit, the presumed active site nucleophile. To define how proteins are degraded, casein was derivatized with fluorescein isothiocyanate to facilitate detection of released products by the proteasome. Many fluorescent peptides were generated, but the relative amounts of different peptides were independent of the duration of the reaction. The rate of disappearance of protein substrates paralleled the rate of appearance of small products. Unlike conventional proteases, proteasome degrades proteins processively without release of polypeptide intermediates. Upon activation by SDS, guanidine, heat (55 degrees C), or partial inhibition with DCI, proteasomes still functioned processively, but generated a different pattern of peptides under each condition. Thus, processivity is an inherent feature of the 20 S proteasome, not requiring all active sites or ATP hydrolysis.
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PMID:Processive degradation of proteins and other catalytic properties of the proteasome from Thermoplasma acidophilum. 899 62

Mutations in the presenilin genes, PS1 and PS2, cause a major portion of early onset familial Alzheimer's disease (FAD). The biological roles of the presenilins and how their pathological mutations confer FAD are unknown. In this study, we set out to examine the processing and degradation pathways of PS2. For regulated expression of PS2, we have established inducible cell lines expressing PS2 under the tight control of the tetracycline-responsive transactivator. Western blot analysis revealed that PS2 was detected as an approximately 53-55-kDa polypeptide (54-kDa PS2) as well as a high molecular mass form (HMW-PS2). Using a stably transfected, inducible cell system, we have found that PS2 is proteolytically cleaved into two stable cellular polypeptides including an approximately 20-kDa C-terminal fragment and an approximately 34-kDa N-terminal fragment. PS2 is polyubiquitinated in vivo, and the degradation of PS2 is inhibited by proteasome inhibitors, N-acetyl-L-leucinal-L-norleucinal and lactacystin. Our studies suggest that PS2 normally undergoes endoproteolytic cleavage and is degraded via the proteasome pathway.
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PMID:Endoproteolytic cleavage and proteasomal degradation of presenilin 2 in transfected cells. 911 Sep 91

The mouse pancreatic beta TC3 and beta TC6-F7 cell lines were used to characterize the effects of interferon-gamma (IFN-y) on beta-cell phenotype and function. Initially, intracellular and secreted insulin were compared in glucose-stimulated cells over time. A significant reduction in insulin content and secretion was observed on a per-cell basis in glucose-stimulated beta TC3 and beta TC6-F7 cells after 12 h of exposure to IFN-gamma. The steadystate level of pre-proinsulin mRNA expression was not affected by IFN-gamma. Thus, we postulate that IFN-gamma's inhibitory actions occur after transcription of pre-proinsulin genes. Time-course analysis of IFN-gamma-regulated mRNA expression of the two intra-MHC-encoded subunits of the proteasome (low-molecular-mass polypeptide [Lmp]-2 and Lmp-7) revealed a correlation between their induction and the inhibitory effects of IFN-gamma on glucose-stimulated insulin production. Increased expression of Lmp-2 and Lmp-7 mRNA was accompanied by a corresponding induction of LMP2 and LMP7 protein expression. Subsequently, major histocompatibility complex (MHC) class I cell-surface expression was significantly increased in IFN-gamma-treated beta TC3 and beta TC6-F7 cells. Exposure of IFN-gamma-treated beta-cells to a peptide aldehyde inhibitor of the proteasome (MG132) significantly attenuated MHC class I cell-surface expression but did not prevent the negative effects of IFN-gamma on glucose responsiveness. Enhanced expression of the MHC class I antigen processing and presentation pathway and diminished insulin production appear to be distinct pathological alterations in beta-cells exposed to the insulitic cytokine IFN-gamma.
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PMID:Interferon-gamma independently activates the MHC class I antigen processing pathway and diminishes glucose responsiveness in pancreatic beta-cell lines. 913 43


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