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)

1. The inhibitory activity of an alkaline protease inhibitor, (Streptomyces subtilisin inhibitor) towards subtilisin is found to decrease by photooxidation sensitized by methylene blue with a clear pH dependence, the midpoint of which is about 6.0. 2. Amino acid analyses of photooxidized Streptomyces subtilisin inhibitor indicate that one of the two histidyl residues and the three methionyl residues are destroyed, concomittant with the loss of inhibitory activity. 3. In accordance with this observation, one of the clearly resolved nuclear magnetic resonances from C2-protons of the two histidyl residues is selectively diminished. This histidyl residue, sensitive to photooxidation and giving a proton magnetic resonance peak at lower field, is assigned to His-106 from peptide analyses. 4. Independent modification of methionyl residues by a reaction with H2O2 or Cl2 also decreases the inhibitory activity of Streptomyces subtilisin inhibitor. 5. Modification of lysyl, tyrosyl and tryptophanyl residues by diazonium-1-H-tetrazole does not lead to the loss of the inhibitory activity. 6. The above results indicate that one or more methionyl residue(s) are essential to the inhibitory activity of Streptomyces subtilisin inhibitor, whereas lysyl, tyrosyl and tryptophanyl residues are not essential to the inhibitory activity. Modification of His-106 is also strongly related to the loss of activity, although its distinct participation in the inactivation mechanism has not been demonstrated.
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PMID:Inactivation of Streptomyces subtilisin inhibitory by chemical modifications. 1 22

In vivo most extracellular iron is bound to transferrin or lactoferrin in such a way as to be unable to catalyze the formation of hydroxyl radical from superoxide (.O2-) and hydrogen peroxide (H2O2). At sites of Pseudomonas aeruginosa infection bacterial and neutrophil products could possibly modify transferrin and/or lactoferrin forming catalytic iron complexes. To examine this possibility, diferrictransferrin and diferriclactoferrin which had been incubated with pseudomonas elastase, pseudomonas alkaline protease, human neutrophil elastase, trypsin, or the myeloperoxidase product HOCl were added to a hypoxanthine/xanthine oxidase .O2-/H2O2 generating system. Hydroxyl radical formation was only detected with pseudomonas elastase treated diferrictransferrin and, to a much lesser extent, diferriclactoferrin. This effect was enhanced by the combination of pseudomonas elastase with other proteases, most prominently neutrophil elastase. Addition of pseudomonas elastase-treated diferrictransferrin to stimulated neutrophils also resulted in hydroxyl radical generation. Incubation of pseudomonas elastase with transferrin which had been selectively iron loaded at either the NH2- or COOH-terminal binding site yielded iron chelates with similar efficacy for hydroxyl radical catalysis. Pseudomonas elastase and HOCl treatment also decreased the ability of apotransferrin to inhibit hydroxyl radical formation by a Fe-NTA supplemented hypoxanthine/xanthine oxidase system. However, apotransferrin could be protected from the effects of HOCl if bicarbonate anion was present during the incubation. Apolactoferrin inhibition of hydroxyl radical generation was unaffected by any of the four proteases or HOCl. Alteration of transferrin by enzymes and oxidants present at sites of pseudomonas and other bacterial infections may increase the potential for local hydroxyl radical generation thereby contributing to tissue injury.
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PMID:Pseudomonas and neutrophil products modify transferrin and lactoferrin to create conditions that favor hydroxyl radical formation. 165 25

In the presence of O2, Fe(III) or Cu(II), and an appropriate electron donor, a number of enzymic and nonenzymic oxygen free radical-generating systems are able to catalyze the oxidative modification of proteins. Whereas random, global modification of many different amino acid residues and extensive fragmentation occurs when proteins are exposed to oxygen radicals produced by high energy radiation, only one or a few amino acid residues are modified and relatively little peptide bond cleavage occurs when proteins are exposed to metal-catalyzed oxidation (MCO) systems. The available evidence indicates that the MCO systems catalyze the reduction of Fe(III) to Fe(II) and of O2 to H2O2 and that these products react at metal-binding sites on the protein to produce active oxygen (free radical?) species (viz; OH, ferryl ion) which attack the side chains of amino acid residues at the metal-binding site. Among other modifications, carbonyl derivatives of some amino acid residues are formed; prolyl and arginyl residues are converted to glutamylsemialdehyde residues, lysyl residues are likely converted to 2-amino-adipylsemialdehyde residues; histidyl residues are converted to asparagine and/or aspartyl residues; prolyl residues are converted to glutamyl or pyroglutamyl residues; methionyl residues are converted to methionylsulfoxide residues; and cysteinyl residues to mixed-disulfide derivatives. The biological significance of these metal ion-catalyzed reactions is highlighted by the demonstration: (i) that oxidative modification of proteins "marks" them for degradation by most common proteases and especially by the cytosolic multicatalytic proteinase from mammalian cells; (ii) protein oxidation contributes substantially to the intracellular pool of catalytically inactive and less active, thermolabile forms of enzymes which accumulate in cells during aging, oxidative stress, and in various pathological states, including premature aging diseases (progeria, Werner's syndrome), muscular dystrophy, rheumatoid arthritis, cataractogenesis, chronic alcohol toxicity, pulmonary emphysema, and during tissue injury provoked by ischemia-reperfusion. Furthermore, the metal ion-catalyzed protein oxidation is the basis of biological mechanisms for regulating changes in enzyme levels in response to shifts from anaerobic to aerobic metabolism, and probably from one nutritional state to another. It is also involved in the killing of bacteria by neutrophils and in the loss of neutrophil function following repeated cycles of respiratory burst activity.
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PMID:Metal ion-catalyzed oxidation of proteins: biochemical mechanism and biological consequences. 228 87

Exposure to various forms of mild oxidative stress significantly increased the intracellular degradation of both "short-lived" and "long-lived," metabolically radiolabeled, cell proteins in cultures of Clone 9 liver cells (normal liver epithelia). The oxidative stresses employed were bolus H2O2 addition; continuous H2O2 flux; the redox cycling quinones, menadione and paraquat; and the aldehydic products of lipid peroxidation, 4-hydroxynonenal, malonyldialdehyde, and hexenal. In general, exposure to more severe oxidative stress produced a concentration-dependent decline in intracellular proteolysis, in some cases to below baseline levels. Oxidatively modified "foreign" proteins (superoxide dismutase and hemoglobin) were also selectively degraded, in comparison with untreated foreign proteins, when added to lysates of Clone 9 liver cells. As with intracellular proteolysis, the degradation of foreign proteins added to cell lysates was greatly increased by mild oxidative modification, but depressed by more severe oxidative modification. The proteinase activity was recovered in > 300-kDa cell fractions, and inhibitor profiles and immunoprecipitation studies indicated that the multicatalytic proteinase complex, proteasome, was responsible for most of the selective degradation observed with mild oxidative stress; up to approximately 95% for intracellular proteolysis and 65-80% for degradation of foreign modified proteins. Seven days of daily treatment with an antisense oligodeoxynucleotide, directed against the initiation codon region of the proteasome C2 subunit gene, severely depressed the intracellular levels of several proteasome subunit polypeptides (by Western blot analysis), and also depressed the H2O2 induced increase in intracellular proteolysis by approximately 95%, without significantly affecting baseline proteolytic rates. Extensive studies revealed only small or no increases in the overall capacity of oxidatively stressed cells to degrade oxidatively modified protein substrates; a finding supported by both Western blot and Northern blot analyses which revealed no significant increase in the levels of proteasome subunit polypeptides or mRNA transcripts. We conclude that mild oxidative stress increases intracellular proteolysis by modifying cellular proteins, thus increasing their proteolytic susceptibility. In contrast, severe oxidative stress diminishes intracellular proteolysis, probably by generating severely damaged cell proteins that cannot be easily degraded (e.g. cross-linked/aggregated proteins), and by damaging proteolytic enzymes. We further conclude that the multicatalytic proteinase complex proteasome is responsible for most of the recognition and selective degradation of oxidatively modified proteins in Clone 9 liver cells.
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PMID:Proteolysis in cultured liver epithelial cells during oxidative stress. Role of the multicatalytic proteinase complex, proteasome. 783 68

The physiologically relevant stress of a flux of H2O2 increased hemoglobin (Hb) degradation in red blood cells (RBC) and increased the proteolytic susceptibility of Hb in vitro. After exposure to low H2O2 flux rates (6-32 microM/min) Hb exhibited increased exposure of hydrophobic (Trp, Met) and basic (Lys) amino acid R groups, increased hydrophobicity, and increased proteolytic susceptibility during subsequent incubation with RBC extracts, a partially purified preparation called Fraction II (which retains all of the proteolytic activities of RBC extracts), or the purified 670-kDa RBC multicatalytic proteinase complex proteasome. Hydrophobicity was measured by butyl-Sepharose hydrophobic interaction chromatography, by the free energy of transfer from water to ethanol, and by heat denaturation assays. Proteolytic susceptibility was measured by release of free alanine, by fluorescamine-reactive free amino groups, and by release of acid-soluble radioactivity from radiolabeled Hb. Low H2O2 flux rates also caused significant charge changes in Hb (isoelectric focusing gels) and extensive noncovalent aggregation (presumably due to increased hydrophobic interactions) but only limited covalent cross-linking (comparison of sodium dodecyl sulfate-polyacylamide gel electrophoresis (SDS-PAGE) and nondenaturing PAGE). Exposure to higher H2O2 flux rates (56-120 microM/min) caused progressive oxidative destruction of exposed hydrophobic amino acids, decreased hydrophobicity as judged by butyl-Sepharose chromatography and heat denaturation assays, increased hydrophilicity as judged by measurements of the free energy of transfer (delta G') from water to ethanol, and decreased proteolytic susceptibility during incubation with RBC extracts, Fraction II, or purified proteasome. High H2O2 flux rates also caused further charge changes and the extensive formation of covalently cross-linked Hb molecules. Linear regression analyses revealed correlations of 0.8-0.99 for the relationship between Hb hydrophobicity and proteolytic susceptibility for both Fraction II and proteasome. Inhibitor studies and SDS activation experiments indicate that proteasome is responsible for most of the Hb degradation during exposure of RBC to H2O2. Previous work yielded essentially identical conclusions for Hb exposed to hydroxyl radicals (R. E. Pacifici, Y. Kono, and K. J. A. Davies, J. Biol. Chem. 268, 15405-15411, 1993). Thus, nonspecific oxidation by .OH and site-specific (metal-catalyzed) oxidation by H2O2 both yield a more hydrophobic Hb molecule with increased proteolytic susceptibility. We propose that increased exposure of hydrophobic, and perhaps basic, amino acids is the general common cause for degradation of oxidized proteins.(ABSTRACT TRUNCATED AT 400 WORDS)
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PMID:Exposure of hydrophobic moieties promotes the selective degradation of hydrogen peroxide-modified hemoglobin by the multicatalytic proteinase complex, proteasome. 820 95

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

Exposure to various forms of oxidative stress (H2O2 and O2.-) significantly increased the intracellular degradation of both "short-lived" and "long-lived" cellular proteins in the human hematopoietic cell line K562. Oxidatively modified hemoglobin and superoxide dismutase used as purified proteolytic substrates were also selectively degraded by K562 cell lysates, but exposure of these protein substrates to very high hydrogen peroxide concentrations actually decreased their proteolytic susceptibility. Our studies found little or no change in the overall capacity of cells and cell lysates to degrade "foreign" oxidized proteins after treatment of K562 cells with hydrogen peroxide or paraquat, a finding supported by proteasome Western blots and unchanged capacity of cell lysates to degrade the fluorogenic peptide succinyl-leucine-leucine-valine-tyrosine-4-methylcoumarin-7-amide. Six days of daily treatment of K562 cells with an antisense oligodeoxynucleotide directed against the initiation codon region of the human proteasome C2 subunit gene dramatically depressed hydrogen peroxide-induced degradation of metabolically radiolabeled intracellular proteins. The actual amount of proteasome in antisense-treated K562 cells was also severely depressed, as revealed by Western blots and by measurements of the degradation of the fluorogenic peptide succinyl-leucine-leucine-valine-tyrosine-4-methylcoumarin-7-amide. The degradation of oxidatively modified foreign protein substrates was also markedly depressed in lysates prepared from K562 cells treated with the proteasome C2 antisense dideoxynucleotide. The inhibitor profile for the degradation of H2O2-modified hemoglobin by K562 cell lysates was consistent with a major role for the ATP-independent 20 S "core" proteasome complex. We conclude that proteasome, probably the 20 S core proteasome complex, is primarily responsible for the selective degradation of oxidatively damaged proteins in human hematopoietic cells. Since "oxidative marking" of cellular proteins by lipoxygenase has been proposed as an important step in red blood cell maturation, it is important to determine which protease or proteases could recognize and degrade such modified substrates. Our results provide evidence that proteasome can, indeed, conduct such selective degradation and appears to be the major cellular protease capable of fulfilling such a role in maturation.
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PMID:Degradation of oxidized proteins in K562 human hematopoietic cells by proteasome. 866 34

It is well established that the functional properties of proteins can be compromised by oxidative damage and, in vivo, proteins modified by oxidants are rapidly degraded. It was hypothesized that oxidants may also affect the ability of proteases to hydrolyze peptides and proteins. We therefore examined the effect of oxidants on the endopeptidase activities of the 650 kDa 20S proteasome or multicatalytic endopeptidase (MCP), which is thought to play a central role in nonlysosomal protein breakdown. Treatment of the MCP with the oxidant system, FeSO4-EDTA-ascorbate, stimulated the peptidase activities of the MCP while H2O2 treatment showed little or no stimulation. However, treatment of the MCP with FeSO4-EDTA-ascorbate or H2O2 stimulated proteinase activity by 480% and 730%, respectively. An endogenous activator of the MCP, PA28, stimulated the acidic, basic, and hydrophobic peptidase activities of the MCP, but had no effect on proteolytic activity. Treatment of PA28 with oxidants in the presence of MCP or alone did not greatly affect PA28's ability to activate the peptidase activities of the MCP. Using nondenaturing polyacrylamide gel electrophoresis, structural alterations in the enzyme which may be responsible for the activation of peptidase and protease activities following exposure to oxidants were investigated. Treatment of the MCP with reagents that activate proteolysis, including H2O2, as well as the serine protease inhibitor 3,4-dichloroisocoumarin and the cysteine protease inhibitor p-(chloromercuri) benzenesulfonic acid, all caused dissociation of the 650 kDa MCP. However, exposure to FeSO4-EDTA-ascorbate resulted in little or no dissociation of the complex. The MCP complex dissociated by p-(chloromercuri) benzenesulfonic acid could be reassociated upon treatment with the reducing agent dithiothreitol, but dithiothreitol failed to completely reassociate 3,4-dichloroisocoumarin- or H2O2 treated MCP. Therefore, chemical modification of the MCP can cause activation with varying degrees of complex dissociation. These results suggest that metabolites, such as reactive oxygen species, in addition to endogenous proteins, such as PA28, are capable of modulating MCP activity.
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PMID:Activation of the multicatalytic endopeptidase by oxidants. Effects on enzyme structure. 867 41

We previously reported cDNA cloning of a novel oxidative stress protein termed A170 from murine macrophages. Further experiments have demonstrated that exposure of the cells to low levels of H2O2 produced by glucose/glucose oxidase markedly induced the 60-kDa A170 protein. This result suggests that the level of A170 protein can also be controlled at posttranscriptional levels, because we showed previously that H2O2 hardly increased the level of A170 mRNA. We have found that proteasome inhibitors markedly induced the A170 protein after 2 to 8 h similarly to glucose/glucose oxidase, suggesting rapid degradation of the A170 protein by proteasome under normal conditions. Activation of cellular signaling pathways either by epidermal growth factor, lipopolysaccharide or tumor necrosis factor-alpha did not enhance the level of the A170 protein. The levels of glucose oxidase-induced A170 protein did not decrease after the addition of cycloheximide. These results suggest that low levels of H2O2 may stabilize the A170 protein, allowing it to accumulate within cells.
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PMID:Low micromolar levels of hydrogen peroxide and proteasome inhibitors induce the 60-kDa A170 stress protein in murine peritoneal macrophages. 912 46

We isolated several species of bacteria from the surface water of a Japanese lake. Of the isolated bacteria, Pseudomonas aeruginosa was the only species which could degrade microcystin LR in vitro. Microcystin LR decreased to 4.5% of the spiked microcystin LR quantity in the P. aeruginosa culture, and was metabolized to (2S, 3S, 8S)-3-amino-2, 6, 8-trimethyl-10-phenyldeca-4E, 6E-dienoic acid (DmADDA). It was possible that P. aeruginosa hydrolysed nucleophilically the peptide bond of microcystin LR. We examined if pyochelin, pyocyanin and alkaline protease produced by P. aeruginosa affected the reduction of microcystin LR. In the result, DmADDA was produced from microcystin LR in the presence of 100 microM H2O2 and 100 microM each of pyochelin, pyocyanin or both. However, the production of DmADDA was slight (2 to 12 mole%). After treatment with P. aeruginosa alkaline protease, DmADDA was produced 75 mole% from microcystin LR. Therefore, we concluded that microcystin was degraded mainly by the action of P. aeruginosa alkaline protease.
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PMID:Microcystin LR degradation by Pseudomonas aeruginosa alkaline protease. 956 41


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