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 wild-type tumor suppressor protein p53 is a short-lived protein that plays important roles in regulation of cell cycle, differentiation, and survival. Mutations that inactivate or alter the tumor suppressor activity of the protein seem to be the most common genetic change in human cancer and are frequently associated with changes in its stability. The ubiquitin system has been implicated in the degradation of p53 both in vivo and in vitro. A mutant cell line that harbors a thermolabile ubiquitin-activating enzyme, E1, fails to degrade p53 at the nonpermissive temperature. Studies in cell-free extracts have shown that covalent attachment of ubiquitin to the protein requires the three conjugating enzymes: E1, a novel species of ubiquitin-carrier protein (ubiquitin-conjugating enzyme; UBC),E2-F1, and an ubiquitin-protein ligase, E3. Recognition of p53 by the ligase is facilitated by formation of a complex between the protein and the human papillomavirus (HPV) oncoprotein E6. Therefore, the ligase has been designated E6-associated protein (E6-AP). However, these in vitro studies have not demonstrated that the conjugates serve as essential intermediates in the proteolytic process. In fact, in many cases, conjugation of ubiquitin to the target protein does not signal its degradation. Thus, it is essential to demonstrate that p53-ubiquitin adducts serve as essential proteolytic intermediates and are recognized and degraded by the 26S protease complex, the proteolytic arm of the ubiquitin pathway. In this study, we demonstrate that conjugates of p53 generated in the presence of purified, E1, E2, E6-AP, E6, ubiquitin and ATP, are specifically recognized by the 26S protease complex and degraded. In contrast, unconjugated p53 remains stable. The ability to reconstitute the system from purified components will enable detailed analysis of the recognition process and the structural motifs involved in targeting the protein for degradation.
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PMID:Complete reconstitution of conjugation and subsequent degradation of the tumor suppressor protein p53 by purified components of the ubiquitin proteolytic system. 803 27

The degradation of cytoplasmic antigens to peptides presented by class I MHC molecules is thought to be mediated by the ubiquitin/proteasome pathway. Support for this view came from our observation that the subunit composition of proteasomes can be changed by interferon-gamma (IFN-gamma) treatment. Thereby two subunits, LMP2 and LMP7, which are encoded in the MHC class II region, are incorporated into the proteasomal complex, whereas other subunits disappear. In the experiments reported in this communication we studied the subunit changes occurring in cell lines where the expression of LMP2 or LMP7 can be regulated individually either by IFN-gamma induction or by applying a new system to control the expression of transfected LMPs. In both situations LMP2 induction leads exclusively to the disappearance of housekeeping subunit 2, whereas LMP7 affects only subunit 10. Subunit 2 was found to be 76% homologous to LMP2. Since incorporation of LMP2 into the proteasomal complex prevents processing of the subunit 2 precursor, we conclude that LMP2 displaces subunit 2 during assembly. Subunit displacement is most likely a general mechanism to modulate the catalytic activity of the proteasomal complex without changing its structure. Furthermore, the controlled incorporation of transfected subunits into the complex offers a new approach to study proteasome function in vivo.
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PMID:Displacement of housekeeping proteasome subunits by MHC-encoded LMPs: a newly discovered mechanism for modulating the multicatalytic proteinase complex. 804 54

Targeting of different cellular proteins for conjugation and subsequent degradation via the ubiquitin pathway involves diverse recognition signals and distinct enzymatic factors. A few proteins are recognized via their N-terminal amino acid residue and conjugated by a ubiquitin-protein ligase that recognizes this residue. Most substrates, including the N alpha-acetylated proteins that constitute the vast majority of cellular proteins, are targeted by different signals and are recognized by yet unknown ligases. We have previously shown that degradation of N-terminally blocked proteins requires a specific factor, designated FH, and that the factor acts along with the 26S protease complex to degrade ubiquitin-conjugated proteins. Here, we demonstrate that FH is the protein synthesis elongation factor EF-1 alpha. (a) Partial sequence analysis reveals 100% identity to EF-1 alpha. (b) Like EF-1 alpha, FH binds to immobilized GTP (or GDP) and can be purified in one step using the corresponding nucleotide for elution. (c) Guanine nucleotides that bind to EF-1 alpha protect the ubiquitin system-related activity of FH from heat inactivation, and nucleotides that do not bind do not exert this effect. (d) EF-Tu, the homologous bacterial elongation factor, can substitute for FH/EF-1 alpha in the proteolytic system. This last finding is of particular interest since the ubiquitin system has not been identified in prokaryotes. The activities of both EF-1 alpha and EF-Tu are strongly and specifically inhibited by ubiquitin-aldehyde, a specific inhibitor of ubiquitin isopeptidases. It appears, therefore, that EF-1 alpha may be involved in releasing ubiquitin from multiubiquitin chains, thus rendering the conjugates susceptible to the action of the 26S protease complex.
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PMID:Protein synthesis elongation factor EF-1 alpha is essential for ubiquitin-dependent degradation of certain N alpha-acetylated proteins and may be substituted for by the bacterial elongation factor EF-Tu. 805 36

1. Proteins in eukaryotic cells are continually degraded and replaced under precise control mechanisms. Although this continual proteolysis may seem wasteful, it serves several important functions: cells selectively degrade proteins with abnormal sequences or conformations, the accumulation of which could be harmful; the rapid degradation of regulatory peptides and enzymes is essential for the control of metabolic pathways and the cell cycle; and the breakdown of proteins in starvation provides amino acids for gluconeogenesis and energy metabolism. 2. Protein breakdown in eukaryotic cells occurs through distinct pathways: A) lysosomal (involves cathepsins B, H, L, etc.); B) Ca(2+)-dependent (involves Ca(2+)-dependent proteases calpains I and II); C) ATP-dependent, that require or not ubiquitin (comprises at least two large cytosolic proteases, UCDEN and proteasome), and D) ATP-independent (it is not known which proteases are involved in this degradative system). Despite recent dramatic progress, the relative contributions of these pathways to the accelerated proteolysis occurring in normal and pathological states is still largely unknown. 3. In order to identify the cellular mechanisms of skeletal muscle atrophy during fasting and diabetes mellitus, we have studied protein turnover in soleus and EDL muscles from control and fasted (for 24 h) or diabetic rats (1, 3, 5 and 10 days after streptozotocin injection). 4. The increase in muscle proteolysis during fasting seems to be attributable to an enhancement of the energy-requiring process. An increase in the ATP-dependent proteolytic pathway was evident 1 day after food restriction and probably accounted for all of the increased proteolysis demonstrated in the EDL muscles. In parallel with the alterations in the ATP-dependent process, an increase in the ubiquitin-mRNA and proteasome subunit-mRNA was detected. 5. In the acute phase of diabetes (1-3 days) there was an activation of Ca(2+)-dependent (soleus and EDL) and ATP-dependent (EDL) pathways. However, after 5 and 10 days of diabetes the activity of these two pathways fell to values even below control ones. No changes in the lysosomal proteolytic system were observed during diabetes. 6. Although appreciable progress has been made in this research, a large number of important questions remain to be answered, and some of them are discussed in the present paper.
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PMID:Regulation of different proteolytic pathways in skeletal muscle in fasting and diabetes mellitus. 808 98

Reagents that inhibit the ubiquitin-proteasome proteolytic pathway in cells have not been available. Peptide aldehydes that inhibit major peptidase activities of the 20S and 26S proteasomes are shown to reduce the degradation of protein and ubiquitinated protein substrates by 26S particles. Unlike inhibitors of lysosomal proteolysis, these compounds inhibit the degradation of not only abnormal and short-lived polypeptides but also long-lived proteins in intact cells. We used these agents to test the importance of the proteasome in antigen presentation. When ovalbumin is introduced into the cytosol of lymphoblasts, these inhibitors block the presentation on MHC class I molecules of an ovalbumin-derived peptide by preventing its proteolytic generation. By preventing peptide production from cell proteins, these inhibitors block the assembly of class I molecules. Therefore, the proteasome catalyzes the degradation of the vast majority of cell proteins and generates most peptides presented on MHC class I molecules.
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PMID:Inhibitors of the proteasome block the degradation of most cell proteins and the generation of peptides presented on MHC class I molecules. 808 44

We demonstrate an essential role for the proteasome complex in two proteolytic processes required for activation of the transcription factor NF-kappa B. The p105 precursor of the p50 subunit of NF-kappa B is processed in vitro by an ATP-dependent process that requires proteasomes and ubiquitin conjugation. The C-terminal region of p105 is rapidly degraded, leaving the N-terminal p50 domain. p105 processing can be blocked in intact cells with inhibitors of the proteasome or in yeast with proteasome mutants. These inhibitors also block the activation of NF-kappa B and the rapid degradation of I kappa B alpha induced by tumor necrosis factor alpha. Thus, the ubiquitin-proteasome pathway functions not only in the complete degradation of polypeptides, but also in the regulated processing of precursors into active proteins.
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PMID:The ubiquitin-proteasome pathway is required for processing the NF-kappa B1 precursor protein and the activation of NF-kappa B. 808 45

The proteasome is a 700-kD multisubunit enzyme complex with several proteolytically active sites. The enzyme complex is involved in both ubiquitin-dependent and -independent protein degradation and may contribute to the processing of antigens presented by major histocompatibility complex (MHC) class I molecules. Here we demonstrate that treatment of mouse fibroblast cells with 20 U interferon gamma (IFN-gamma) for 3 d induces a change in the proteasome subunit composition and that the beta-type subunit LMP2, which is encoded in the MHC class II region, is incorporated into the enzyme complex. This is paralleled by reduction of the homologous delta-subunit. IFN-gamma stimulation results in a downregulation of the chymotrypsin-like Suc-LLVY-MCA peptide hydrolyzing activity of 20S proteasomes whereas the trypsin-like activity remains unaffected. When tested as a substrate a synthetic 25-mer polypeptide whose sequence covers the antigenic nonapeptide YPHFMPTNL of the MCMV pp89, 20S proteasomes of IFN-gamma-induced cells exhibit altered chymotrypsin-like cleavage site preferences. In the absence of IFN-gamma induction, the naturally processed nonamer peptide that is presented by MHC class I molecules appears as a minor cleavage product. IFN-gamma activation does not result in an increase of the final peptide but results in a different set of peptides. We hypothesize that these peptides represent precursor peptides that can be trimmed to final peptide size.
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PMID:Interferon gamma stimulation modulates the proteolytic activity and cleavage site preference of 20S mouse proteasomes. 811 82

The 26 S proteolytic complex ("26 S proteasome") is a macromolecular assembly thought to be involved in ATP- and ubiquitin-dependent protein degradation in the cytoplasm of higher eukaryotic cells. This complex is composed of one 20 S cylinder particle (multicatalytic proteinase, 20 S proteasome) and two cap-shaped 19 S particles comprising a set of polypeptides in the M(r) range of 35,000-110,000. Here we show that cell supernatant fractions contain both these two subunit complexes as distinct particles as well as assembled to 26 S proteasomes. We have separated and purified all three forms from Xenopus laevis oocytes and have determined their peptidase and protease activities. Using various antibodies specific for either a constitutive p52 polypeptide of the 19 S cap complex or for proteins of the 20 S cylinder particle, we have immunolocalized these complexes in both the cytoplasm and the nucleus of diverse species and cell types. The occurrence of all three forms, the 26 S proteasome, the 20 S cylinder particle, and the 19 S cap complex in the nucleoplasm has also been demonstrated in analyses of isolated giant nuclei from Xenopus oocytes. In addition, we show that the 19 S and 20 S subcomplexes can be released from 26 S proteasomes by ATP depletion and that readdition of ATP to 19 S and 20 S particles in cell extracts leads to the reformation of 26 S proteasomes. We discuss that all three particles (19 S, 20 S, and 26 S) exist in a dynamic equilibrium in both cell compartments and serve cytoplasmic as well as nucleus-specific functions.
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PMID:Distinct 19 S and 20 S subcomplexes of the 26 S proteasome and their distribution in the nucleus and the cytoplasm. 812 97

The 240-kDa proteasome inhibitor has been reported to be an ATP-stabilized component (CF-2) of the 26 S proteasome complex. We now report that this inhibitory factor is indistinguishable from delta-aminolevulinic acid dehydratase (ALAD), the second enzyme in the pathway of heme synthesis, based upon the following observations: 1) common sequence of the first 14 N-terminal amino acids; 2) identical migration on native and SDS-polyacrylamide gel electrophoresis; 3) identical isoelectric points of pH 7.1; 4) cross-reactivity of specific polyclonal antibodies; 5) similar dehydratase and proteasome inhibitor specific activities in both proteins; and 6) the presence of both activities in recombinant ALAD. The dual role of this protein as CF-2 in the ATP/ubiquitin-dependent pathway and in heme synthesis may be an example of "gene sharing" and explains the unexpected abundance of ALAD noted in earlier studies.
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PMID:240-kDa proteasome inhibitor (CF-2) is identical to delta-aminolevulinic acid dehydratase. 817 43

Metabolic acidosis often leads to loss of body protein due mainly to accelerated protein breakdown in muscle. To identify which proteolytic pathway is activated, we measured protein degradation in incubated epitrochlearis muscles from acidotic (NH4Cl-treated) and pair-fed rats under conditions that block different proteolytic systems. Inhibiting lysosomal and calcium-activated proteases did not reduce the acidosis-induced increase in muscle proteolysis. However, when ATP production was also blocked, proteolysis fell to the same low level in muscles of acidotic and control rats. Acidosis, therefore, stimulates selectively an ATP-dependent, nonlysosomal, proteolytic process. We also examined whether the activated pathway involves ubiquitin and proteasomes (multicatalytic proteinases). Acidosis was associated with a 2.5- to 4-fold increase in ubiquitin mRNA in muscle. There was no increase in muscle heat shock protein 70 mRNA or in kidney ubiquitin mRNA, suggesting specificity of the response. Ubiquitin mRNA in muscle returned to control levels within 24 h after cessation of acidosis. mRNA for subunits of the proteasome (C2 and C3) in muscle were also increased 4-fold and 2.5-fold, respectively, with acidosis; mRNA for cathepsin B did not change. These results are consistent with, but do not prove that acidosis stimulates muscle proteolysis by activating the ATP-ubiquitin-proteasome-dependent, proteolytic pathway.
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PMID:Metabolic acidosis stimulates muscle protein degradation by activating the adenosine triphosphate-dependent pathway involving ubiquitin and proteasomes. 818 44


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