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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)

Eukaryotic proteasomes are multicatalytic proteinase complexes with a molecular weight of 750 kDa, containing, respectively, two copies of a hetero-heptamer of alpha-type subunits and one of beta-type subunits, (alpha 1-7 beta 1-7)2. Proteasome was purified from bovine liver and crystallized into a hexagonal system with cell dimensions of a = b = 121.83(2) A, c = 930.68(6) A. A cylindrical particle size of 122 A diameter and 155 A height was determined from the molecular packing in a unit cell. The crystal gave diffraction spots up to at least 4.4 A resolution, which was the minimum spacing of the camera used. The overall temperature factor of the enzyme was estimated to be in the range of 36.2 to 25.8 A2. These results imply that the enzyme complex has a unique ordered structure comprising multisubunits with two types of hetero-heptamer. This ordered structure may facilitate highly organized cooperation of individual functions of subunits within the enzyme complex.
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PMID:Ordered structure of the crystallized bovine 20S proteasome. 762 9

The function of the proteasome is controlled by a variety of specific regulatory proteins including activators, inhibitors, and modulators. Two recently discovered activators, termed PA28 and PA700, bind to the terminal rings of the proteasome to form proteasome-regulatory complexes which display greatly increased proteolytic activity. PA28 is a high-affinity activator of the proteasome's multiple peptidase activities. The carboxyl terminus of PA28 is required for its binding to the proteasome. PA700 binds to the proteasome via an ATP-dependent mechanism. PA700 has ATPase activity, and at least four of PA700's 16 subunits are members of a protein family containing a concensus sequence for ATP binding. Proteasome-PA700 complexes are activated with respect to both the hydrolysis of peptide substrates and the hydrolysis of ubiquitinated proteins.
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PMID:Regulatory proteins of the proteasome. 769 29

Proteasomes are intricate cellular proteases that are able to degrade many protein and peptide substrates in vitro. These particles are structurally complex; they are assembled from at least 14 small molecular mass polypeptide subunits to form mature 20S proteasomes. Recently, we demonstrated that proteasome subsets may be discriminated by serological criteria, and have found that subtle differences in the subunit composition of proteasomes can alter their cleavage specificity. Proteasome structural complexity is further enhanced when some proteasomes associate with additional proteins to form a 26S ATP- and ubiquitin-dependent protease. Herein we confirm the existence of distinct cellular forms of proteasomes, and show that they differ in their hydrophobic characteristics. We have reproducibly purified, using solely biochemical techniques, distinct proteasome subsets similar to the serologically defined LMP2+ and LMP2- proteasomes. These proteasome subsets differ in their expression of at least three polypeptides, and both lack several additional polypeptides as compared to the serologically defined LMP2+ and LMP2- proteasomes. Finally, we demonstrate that these structurally unique proteasomes differ in their capacity to cleave a defined panel of fluorogenic peptide substrates. It appears that mammalian cells might recruit and modify proteasomes to perform distinct cellular tasks.
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PMID:Biochemical purification of distinct proteasome subsets. 769 32

We examined the effects of a synthetic glucocorticoid (dexamethasone; Dex) on protoeolysis and on protease messenger RNA (mRNA) concentrations in rat L8 skeletal myotube cultures. Protein degradation was measured as release of radioactive trichloroacetic acid-soluble material from intracellular proteins pre-labelled with [3H]tyrosine. Dex (1 microM) stimulated protein degradation (P < 0.01). This effect was entirely blocked by the glucocorticoid antagonist, RU38486 (mifepristone; P < 0.01). Hence, actions of Dex on muscle protein degradation are mediated via intracellular glucocorticoid receptors. Molecular mechanisms by which glucocorticoids stimulate protein degradation in skeletal muscle are not known. Here, we investigated the regulation of protease (cathepsin B, cathepsin D, proteasome C2 subunit and m-calpain) mRNA concentrations by Dex in cultured L8 muscle cells. Cathepsin B mRNA concentration was enhanced 3.3-fold by Dex. This effect was blocked by RU38486. RU38486 alone did not affect cathepsin B mRNA concentration or mRNAs of other proteases. Concentrations of cathepsin D and m-calpain mRNAs were also increased by Dex. These effects were also abolished by RU38486. Proteasome C2 mRNA was unaffected by Dex and Dex reduced alpha-tubulin mRNA. Thus, glucocorticoids specifically regulate the concentrations of mRNAs encoding some proteases in muscle cells. The regulation of protease mRNA concentration is mediated via interaction between Dex with glucocorticoid receptors and is independent of the actions of Dex on mRNA encoding house-keeping proteins. These changes may underlie glucocorticoid-dependent control of proteolysis in muscle.
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PMID:Effects of dexamethasone on protein degradation and protease gene expression in rat L8 myotube cultures. 775 36

We examined the effects of horse and fetal bovine sera and insulin-like growth factor I (IGF-I) on proteolysis and protease gene expression in rat L8 skeletal myotube cultures. Protein degradation was measured as release of radioactive trichloroacetic acid (TCA)-soluble materials from intracellular proteins prelabeled with [3H]tyrosine. Horse serum and fetal bovine serum inhibited (P < .05) protein degradation by 19.7 and 8.1%, respectively. The IGF-I at 200 ng/mL inhibited protein degradation by 14% (P < .01) over a 6-h measurement period. To study the regulation of proteolysis by IGF-I, we evaluated its effects on protease mRNA and alpha-tubulin mRNA concentrations by Northern blot analysis. Proteases under investigation included cathepsins B and D, proteasome C2 subunit, and m-calpain. The IGF-I had no effect (P > .05) on cathepsin B and D gene expression but slightly increased (P < .05) m-calpain and alpha-tubulin mRNA concentrations. Proteasome mRNA concentration was reduced (P < .05) by IGF-I treatment. The changes in proteasome mRNA levels paralleled the IGF-I-dependent alterations in proteolysis. These observations suggest that effects of IGF-I on muscle protein degradation may be mediated by the specific down-regulation of proteasomal subunit mRNAs.
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PMID:Effects of serum and insulin-like growth factor I on protein degradation and protease gene expression in rat L8 myotubes. 800 47

Eukaryotic cells contain a major intracellular proteolytic activity known as the proteasome. The proteasome is a strongly conserved cylindrical structure of high molecular weight (650 kDa, approximately 20 S) and demonstrates multiple endopeptidase activities. The general structural, biochemical and genetic features of the proteasome are conserved from archaebacteria through yeast to humans. This structure fulfills an essential role by functioning as the proteolytic core of a 26 S multienzyme complex responsible for the energy-dependent degradation of ubiquitinated proteins. The bulk of intracellular proteolysis appears to be through the ubiquitin-dependent pathway. Incorporation of the proteasome into the 26 S multienzyme complex appears to confer both a specificity for ubiquitinated proteins as well as a means to tightly regulate proteolytic activity. Thus, one function of the proteasome is required for the degradation of either abnormal or certain regulatory proteins by the ubiquitin pathway. Proteasome subunits appear to be encoded by a related gene family as defined by extensive sequence similarities. The gene products are confined to either of two general classes: alpha-type which appear to be structural and beta-type which may be catalytic. Genes encoding at least two proteasome subunits map to the Major Histocompatibility Complex. Accumulating evidence points to the proteasome (or a specialized form) participating in the cytosolic degradation of these viral proteins upon cellular infection.(ABSTRACT TRUNCATED AT 250 WORDS)
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PMID:The role of the proteasome in cellular protein degradation. 800 15

Proteasome, a large protein complex with ATP-dependent protease activities, is composed of non-identical but closely related multi-subunits. Using cDNAs for rat proteasome subunits as probes, we obtained three cDNA clones for two Xenopus proteasome subunits from ovary cDNA library. The primary structures of the three cDNAs showed high homology to the corresponding proteasome subunits of other mammalian species (above 90%) and also considerable homology to those of Drosophila and yeast. These results indicate that the sequences of proteasome subunits are well conserved during evolution. Northern blot hybridization revealed that RNAs for the newly isolated subunits (XC8 and XC9) and the previously isolated subunit (XC3) occur at very high levels in testis and ovary, at moderately high levels in lung, skin kidney and spleen, and at low levels in liver, stomach and muscle. It was also shown that relative amounts of the mRNAs for the three subunits are similar in all the adult tissues examined. From these results, we concluded that the expression of the genes for the three subunits (XC3 XC8 and XC9-1) takes place in a roughly coordinated manner in different adult tissues.
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PMID:Molecular cloning of cDNAs for two Xenopus proteasome subunits and their expression in adult tissues. 821 17

Cells exposed to oxidative stress have been shown previously to exhibit both protein oxidation and increased proteolysis. Experiments conducted with purified proteins in vitro have indicated that oxidatively modified proteins may be selectively degraded by intracellular proteases, but a definitive cause-and-effect relationship has not been demonstrated previously in intact cells. Several investigators have proposed that oxidatively modified proteins are selectively degraded within cells, but the possibility that oxidants may activate intracellular proteases (directly or indirectly) to catalyze the indiscriminate degradation of undamaged proteins has not been discounted. Armed with the knowledge that dityrosine is a specific product of protein oxidation, we undertook a series of experiments to test the hypothesis that oxidized proteins undergo selective intracellular degradation. Our results demonstrate that dityrosine is produced in the hemoglobin molecule when red blood cells are exposed to a continuous flux of hydrogen peroxide (H2O2). The dityrosine so produced is only released from the hemoglobin by proteolysis and is stable to prolonged incubation with cell extracts. Inhibitors of proteolysis have no effect on dityrosine production but do effectively prevent dityrosine release. Proteasome (the 670-kDa multicatalytic proteinase complex, that we have previously called macroxyproteinase or MOP (Pacifici, R. E., Salo, D. C., and Davies, K. J. A. (1989) Free Radical Biol. & Med. 7, 521-526; Salo, D. C., Pacifici, R. E., Lin, S. W., Giulivi, C., and Davies, K. J. A. (1990) J. Biol. Chem. 265, 11919-11927; Pacifici, R. E., and Davies, K. J. A. (1991) Gerontology 37, 166-180) appears responsible for dityrosine release during the selective degradation of oxidatively modified proteins in red blood cells and red cell extracts. We conclude that the elevated rates of proteolysis observed in response to oxidative stress do, indeed, reflect selective degradation of oxidatively modified (damaged) proteins. Despite a relatively low production rate, dityrosine has a high fluorometric quantum yield and is, of course, a specific product of protein oxidation. As an apparently stable metabolic end product, dityrosine may prove to be an extremely valuable (cellular or urinary) marker or index of organismal oxidative stress.
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PMID:Dityrosine and tyrosine oxidation products are endogenous markers for the selective proteolysis of oxidatively modified red blood cell hemoglobin by (the 19 S) proteasome. 847 19

Proteasome subunits are encoded by members of the same gene family and can be divided into two groups based on their similarity to the alpha and beta subunits of the simpler proteasome isolated from Thermoplasma acidophilum. RN3 is the beta-type subunit, N3, of rat proteasomes which has been implicated in the peptidylglutamyl-peptide hydrolase activity of the proteinase complex. We have expressed recombinant RN3 protein in Escherichia coli in order to raise subunit-specific polyclonal antibodies. Identification of the position of RN3 on two-dimensional PAGE gels of purified rat liver proteasomes showed a single protein spot of molecular mass 24 kDa and of pI value of about 5. This protein has a free N-terminus, having undergone post-translational processing. After immunoprecipitation from [35S]methionine-labelled human embryo lung L-132 cells using anti-RN3 antibodies, two radiolabelled spots were observed on two-dimensional PAGE gels, one corresponding to the mature N3, the other of molecular mass 28.5 kDa and pI value around 5, which was probably the unprocessed form of N3. However, the latter protein had a higher molecular mass (31 kDa) than was predicted from the sequence of previously cloned cDNA. Therefore rapid amplification of cDNA ends ("RACE') was carried out to determine the full sequence. The lack of detectable RN3 precursor in purified rat liver proteasomes suggests that the processing probably accompanies assembly of the complex. The half-life of the processing was determined to be 31 min in growing L-132 cells. The unprocessed form of N3 was not observed after immunoprecipitation of 35S-labelled complexes with anti-proteasome antibodies. There was no evidence to suggest that unprocessed N3 is found in precursor complexes which have been implicated in the assembly of some other unprocessed beta-type subunits. Interestingly also, the site of cleavage of N3 (ITR decreases TQN) differs significantly from those of other processed animal beta-type proteasome subunits [(H/T)G decreases TT(T/L)], many of which resemble more closely the cleavage site of the Thermoplasma acidophilum beta subunit.
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PMID:Processing of N3, a mammalian proteasome beta-type subunit. 864 51

Proteasome-dependent degradation of regulatory proteins is a known mechanism of cell cycle control. We found that the proteasome-specific inhibitor lactacystin (LC) induced expression of the cell cycle inhibitor p21WAF1/CIP1 in human cancer cells regardless of their p53 status. Both wild-type (wt) p53 and p21 protein levels increased by two hours in wt p53 containing cells, whereas mutant (mt) p53 levels decreased and the increase in p21 levels was delayed to 6 hr following inhibition of proteolysis by LC in mt p53 expressing cells. We found that wt but not mt p53 expressing cells increased p21 mRNA and p21-promoter reporter levels following LC exposure, suggesting transcriptional induction of p21. Inhibition of protein synthesis by cycloheximide demonstrated increased p21 protein half-life in the presence of LC in mutant p53 containing cells. p21 induction was correlated with the cytostatic effects of LC. The results suggest that p21 protein expression could be increased by transcriptional mechanisms as well as inhibition of proteolysis by LC.
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PMID:Proteasome-dependent regulation of p21WAF1/CIP1 expression. 887 53


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