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)

We examined changes in proteinase activities in P19 embryonal carcinoma cells during retinoic acid-induced differentiation. The interleukin-1 beta converting enzyme (ICE)-like Ac-YVAD-MCA hydrolytic activity was increased about 6-fold by treatment with retinoic acid. This activity was inhibited by N-ethylmaleimide and Ac-YVAD-H but not by E-64, EDTA, PMSF, or amastatin. The ICE-like activity in P19 cells eluted as a single peak just after the void volume on gel filtration. No ICE-like activity was observed at a molecular mass of 30-50 kDa. Enzymatic purification, Western blot analysis, and an immunoabsorption study demonstrated that the ICE-like activity in P19 cells is caused by the proteasome, and is stimulated during retinoic acid-induced differentiation. The proteasome purified from mouse liver also cleaved Ac-YVAD-MCA. These results strongly suggest that the proteasome is a major ICE-like proteinase in P19 cells and may be involved in the neural differentiation and the apoptotic pathway.
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PMID:Identification of an interleukin-1 beta converting enzyme-like activity that increases upon treatment of P19 cells with retinoic acid as the proteasome. 894 29

The development of small molecule peptide-based activators of the 20S proteasome or multicatalytic proteinase complex was initiated. The enhancement of antigen presentation by transfection of the protein activator PA28alpha into a mouse fibroblast cell line [10] supports the potential use of small molecule activators in stimulating the immune response. Four classes of peptide-based activators were synthesized, i.e. peptidyl alcohols, esters, p-nitroanilides and nitriles. These compounds markedly and reversibly stimulated the hydrolysis of suc-LLVY-MCA, Z-LLE-NA and Z-GPALG-p-aminobenzoate as well as hydrolysis of the decapeptide angiotensin I. Stimulation was due to a decrease in the Km and increase in the Vmax of the substrate. In general, the EC50 for activation ranged from 50-150 mM and maximal stimulation varied from 3 to 15 fold depending on the activity measured. Z-IE(Ot-Bu)AL-p-nitroanilide, a proteasome substrate, markedly stimulated the hydrolysis of Z-GPALG-pAB by binding to a saturable high affinity site distinct from its binding site as substrate. Since all effective activators contain hydrophobic groups in positions P1-P5, low aqueous solubility is a limitation of these compounds. Competition experiments suggest that these activators bind to the same site as PA28.
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PMID:Synthetic peptide-based activators of the proteasome. 922 92

The PA700-like proteasome activator complex was highly purified from porcine erythrocytes, and its properties were compared with those of the regulatory complex disassembled from the purified 26S proteasome. The molecular mass of the PA700-like complex, which comprises 25-110-kDa subunits, was estimated to be 800 kDa by Superose 6 gel filtration. This complex showed neither ATPase activity nor peptidase activity toward Suc-Leu-Leu-Val-Tyr-MCA. Nevertheless, it was possible to make a high molecular mass complex from the purified PA700-like complex by incubating with the 20S proteasome in the presence of ATP. In contrast, the regulatory complex dissociated from the 26S proteasome did not reconstitute a larger complex under the same conditions. The subunit composition of the PA700-like complex was similar but not identical to that of the regulator complex dissociated from the 26S proteasome: the former complex had a 25-kDa subunit which is absent in the latter, whereas the latter had two or three 43-kDa subunits lacking in the former. These results indicate that the purified PA700-like proteasome activator complex is structurally and functionally distinct from the regulatory complex dissociated from the 26S proteasome, implying the involvement of modulating factors in the 26S proteasome assembly.
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PMID:Difference between PA700-like proteasome activator complex and the regulatory complex dissociated from the 26S proteasome implies the involvement of modulating factors in the 26S proteasome assembly. 927 59

Full-length cDNAs for three human proteasome activator subunits, called REGalpha, REGbeta, and REGgamma, have been expressed in Escherichia coli, and the purified recombinant proteins have been characterized. Recombinant alpha or gamma subunits form heptameric species; recombinant beta subunits are found largely as monomers or small multimers. Each recombinant REG stimulates cleavage of fluorogenic peptides by human red cell proteasomes. The pattern of activated peptide hydrolysis is virtually identical for REGalpha and REGbeta. These two subunits, alone or in combination, stimulate cleavage after basic, acidic, and most hydrophobic residues in many peptides. Recombinant alpha and beta subunits bind each other with high affinity, and the REGalpha/beta heteromeric complex activates hydrolysis of LLVY-methylcoumaryl-7-amide (LLVY-MCA) and LLE-beta-nitroanilide (LLE-betaNA) more than REGalpha or REGbeta alone. Using filter binding and gel filtration assays, recombinant REGgamma subunits were shown to bind themselves but not alpha or beta subunits. REGgamma differs from REGalpha and REGbeta in that it markedly stimulates hydrolysis of peptides with basic residues in the P1 position but only modestly activates cleavage of LLVY-MCA or LLE-betaNA by the proteasome. REGgamma binds the proteasome with higher affinity than REGalpha or REGbeta yet with lower affinity than complexes containing both REGalpha and REGbeta. In summary, each of the three REG homologs is a proteasome activator with unique biochemical properties.
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PMID:Characterization of recombinant REGalpha, REGbeta, and REGgamma proteasome activators. 932 61

When an effective concentration of doxorubicin (DXR) was added into L1210 of a mouse leukemia cell line, DXR was rapidly distributed much more in the nuclei than in the other organelle within a few minutes. A [14C]DXR-binding fraction was obtained from the cytosol prepared from L1210 cells. The fraction was adsorbed to hydroxylapatite matrix and eluted from the matrix by 50-150 mM potassium phosphate buffer. The fraction showed high DXR-binding and Suc-Leu-Leu-Val-Tyr-MCA-degrading activity. The binding of [14C]DXR was inhibited by unlabeled DXR. Gel chromatography of the fraction with Sephacryl S-300 separated two fractions of high molecular weight (Peak I, approx. 750 kDa) and low molecular weight (Peak II). Peak I showed proteolytic activity. [14C]DXR-binding Peak I had much higher affinity to DNA-cellulose than [14C]DXR-binding Peak II. [14C]DXR-Peak I complex also was retained into the nuclei isolated from L1210 cells, temperature-dependently. These results suggest that a specific carrier to translocate DXR from cytoplasm into nucleus exists in L1210 cell and the carrier is proteasome.
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PMID:Proteasome is a carrier to translocate doxorubicin from cytoplasm into nucleus. 960 Mar 27

Heat-shock protein 90 (Hsp 90) has been implicated in both protection against oxidative inactivation and inhibition of the multicatalytic proteinase (MCP, also known as 20 S proteasome). We report here that the protective and inhibitory effects of Hsp 90 depend on the activation state of the proteasome. Hsp 90 (and also alpha-crystallin) inhibits the N-Cbz-Leu-Leu-Leu-MCA-hydrolysing activity (Cbz=benzyloxycarbonyl; MCA=7-amido-4-methylcoumarin) when the rat liver MCP is in its latent form, but no inhibitory effects are observed when the MCP is in its active form. Metal-catalysed oxidation of the active MCP inactivates the Ala-Ala-Phe-MCA-hydrolysing (chymotrypsin-like), N-Boc-Leu-Ser-Thr-Arg-MCA-hydrolysing (trypsin-like; Boc=t-butyloxycarbonyl), N-Cbz-Leu-Leu-Glu-beta-naphthylamine-hydrolysing (peptidylglutamyl-peptide hydrolase) and N-Cbz-Leu-Leu-Leu-MCA-hydrolysing activities, whereas these activities are actually increased when the MCP is in its latent form. Hsp 90 protects against oxidative inactivation of the trypsin-like and N-Cbz-Leu-Leu-Leu-MCA-hydrolysing activities of the MCP active form, and alpha-crystallin protects the trypsin-like activity. The specificity of the Hsp 90-mediated protection was assessed by a quantitative analysis of the two-dimensional electrophoretic pattern of MCP subunits before and after oxidation of the MCP, in the presence or absence of Hsp 90. Treatment of the FAO hepatoma cell line with iron and ascorbate was found to inactivate the MCP. Hsp 90 overexpression obtained by challenging the cells with iron was associated with a decreased susceptibility to oxidative inactivation of the MCP trypsin-like activity. Depletion of Hsp 90 by using antisense oligonucleotides resulted in an increased susceptibility to oxidative inactivation of the MCP trypsin-like activity, providing evidence for the physiological relevance of Hsp 90-mediated protection of the MCP.
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PMID:Protection from oxidative inactivation of the 20S proteasome by heat-shock protein 90. 965 82

Sperm proteasomes are thought to be involved in sperm binding to and in sperm penetration through the vitelline coat of the eggs of the stolidobranch ascidian Halocynthia roretzi. However, it is not known whether they are involved in the fertilization of eggs of other ascidians. Therefore, we investigated whether sperm proteasomes are also involved in the fertilization of the eggs of the primitive phlebobranch ascidian Ciona intestinalis. Fertilization of the eggs of C. intestinalis was potently inhibited by the proteasome inhibitors MG115 and MG132 but not by the cysteine protease inhibitor E-64-d. On the other hand, neither fertilization of the vitelline coat-free eggs nor sperm binding to the vitelline coat was inhibited by the two proteasome inhibitors at a concentration sufficient to inhibit fertilization of intact eggs. These results indicate that the proteasome plays an essential role in sperm penetration through the vitelline coat rather than in sperm binding to the coat or in sperm-egg membrane fusion. The proteasome activity, which was detected in the sperm extract using Suc-Leu-Leu-Val-Tyr-MCA as a substrate, was strongly inhibited by both MG115 and MG132, and was weakly inhibited by chymostatin, whereas neither leupeptin nor E-64-d inhibited the activity. The molecular mass of the enzyme was estimated to be 600-kDa by Superose 12 gel filtration, and the activity in sperm extract was immunoprecipitated with an anti-proteasome antibody. These results indicate that the proteasome present in sperm of C. intestinalis is involved in fertilization, especially in the process of sperm penetration through the vitelline coat, probably functioning as a lysin.
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PMID:Participation of sperm proteasome in fertilization of the phlebobranch ascidian Ciona intestinalis. 966 33

Oxidatively modified ferritin is selectively recognized and degraded by the 20S proteasome. Concentrations of hydrogen peroxide (H2O2) higher than 10 micromol/mg of protein are able to prevent proteolytic degradation. Exposure of the protease to high amounts of oxidants (H2O2, peroxynitrite and hypochlorite) inhibits the enzymic activity of the 20S proteasome towards the fluorogenic peptide succinyl-leucine-leucine-valine-tyrosine-methylcoumarylamide (Suc-LLVY-MCA), as well as the proteolytic degradation of normal and oxidant-treated ferritin. Fifty per cent inhibition of the degradation of the protein substrates was achieved using 40 micromol of H2O2/mg of proteasome. No change in the composition of the enzyme was revealed by electrophoretic analysis up to concentrations of 120 micromol of H2O2/mg of proteasome. In further experiments, it was found that the 26S proteasome, the ATP- and ubiquitin-dependent form of the proteasomal system, is much more susceptible to oxidative stress. Whereas degradation of the fluorogenic peptide, Suc-LLVY-MCA, by the 20S proteasome was inhibited by 50% with 12 micromol of H2O2/mg, 3 micromol of H2O2/mg was enough to inhibit ATP-stimulated degradation by the 26S proteasome by 50%. This loss in activity could be followed by the loss of band intensity in the non-denaturing gel. Therefore we concluded that the 20S proteasome was more resistant to oxidative stress than the ATP- and ubiquitin-dependent 26S proteasome. Furthermore, we investigated the activity of both proteases in K562 cells after H2O2 treatment. Lysates from K562 cells are able to degrade oxidized ferritin at a higher rate than non-oxidized ferritin, in an ATP-independent manner. This effect could be followed even after treatment of the cells with H2O2 up to a concentration of 2mM. The lactacystin-sensitive ATP-stimulated degradation of the fluorogenic peptide Suc-LLVY-MCA declined, after treatment of the cells with 1mM H2O2, to the same level as that obtained without ATP stimulation. Therefore, we conclude that the regulation of the 20S proteasome by various regulators takes place during oxidative stress. This provides further evidence for the role of the 20S proteasome in the secondary antioxidative defences of mammalian cells.
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PMID:Comparative resistance of the 20S and 26S proteasome to oxidative stress. 979 5

Changes in proteasome activities were observed during starfish oocyte maturation induced by a maturation-inducing hormone, 1-methyladenine. Succinyl-Leu-Leu-Val-Tyr-MCA-hydrolyzing proteasome activity in immature oocytes showed a main peak of a 1500-kDa fraction and a shoulder centered at a 650-kDa fraction on Superose 6 gel-filtration chromatography in the presence of ATP and glycerole. The 1500-kDa activity transiently decreased and then increased at about a half the time required for germinal vesicle breakdown (GVBD). In contrast, the 650-kDa activity showed only a slight change during the maturation process. The activity of the 1500-kDa complex, unlike that of the 650-kDa complex, was immunoprecipitated with an antibody raised against regulatory subunits of mammalian 26S proteasomes, whereas both 1500- and 650-kDa activities were immunoprecipitated with anti-20S proteasome antibody. In addition, the 1500-kDa complex showed an ATP/ubiquitin-dependent proteolytic activity. These results indicate that the 1500- and 650-kDa complexes correspond to the mammalian 26S and 20S proteasomes, respectively. Immunoblot analysis revealed that the change in the 26S proteasomal activity is due to the change in the amount of the 20S proteasome subcomplex. Taken together, the proteasome undergoes changes in molecular assembly and activities during hormone-induced oocyte maturation.
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PMID:The 26S proteasome assembly is regulated by a maturation-inducing hormone in starfish oocytes. 991 40

The 20S proteasome has been shown to be largely responsible for the degradation of oxidatively modified proteins in the cytoplasm. Nuclear proteins are also subject to oxidation, and the nucleus of mammalian cells contains proteasome. In human beings, tumor cells frequently are subjected to oxidation as a consequence of antitumor chemotherapy, and K562 human myelogenous leukemia cells have a higher nuclear proteasome activity than do nonmalignant cells. Adaptation to oxidative stress appears to be one element in the development of long-term resistance to many chemotherapeutic drugs and the mechanisms of inducible tumor resistance to oxidation are of obvious importance. After hydrogen peroxide treatment of K562 cells, degradation of the model proteasome peptide substrate suc-LLVY-MCA and degradation of oxidized histones in nuclei increases significantly within minutes. Both increased proteolytic susceptibility of the histone substrates (caused by modification by oxidation) and activation of the proteasome enzyme complex occur independently during oxidative stress. This rapid up-regulation of 20S proteasome activity is accompanied by, and depends on, poly-ADP ribosylation of the proteasome, as shown by inhibitor experiments, 14C-ADP ribose incorporation assays, immunoblotting, in vitro reconstitution experiments, and immunoprecipitation of (activated) proteasome with anti-poly-ADP ribose polymerase antibodies. The poly-ADP ribosylation-mediated activated nuclear 20S proteasome is able to remove oxidatively damaged histones more efficiently and therefore is proposed as an oxidant-stimulatable defense or repair system of the nucleus in K562 leukemia cells.
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PMID:Poly-ADP ribose polymerase activates nuclear proteasome to degrade oxidatively damaged histones. 1033 69


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