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)

Yarrowia lipolytica DO613, carrying the xpr6-13 mutation, secretes an inactive precursor of alkaline extracellular protease that has not been cleaved after the Lys-Arg at the end of the pro-region. Compared to wild type, DO613 membrane preparations had significantly reduced ability to cleave after Lys-Arg of an artificial substrate. The XPR6 gene was cloned by complementation by screening for restoration of production of alkaline protease activity. Sequencing of a 3735 base pair SalI-SphI XPR6 fragment revealed a large open reading frame with a coding capacity of 976 amino acids (molecular weight, 110,016). The deduced amino acid sequence had significant homology to Saccharomyces cerevisiae Kex2p, a processing endoprotease that cleaves after pairs of basic amino acids. Disruption of the XPR6 gene was not lethal, but it resulted in several phenotypic changes. First, essentially no mature alkaline extracellular protease was produced indicating that the low levels produced by strains carrying previously isolated xpr6 alleles were due to leaky mutations. Second, mating type B strains carrying the disrupted XPR6 gene did not mate, but mating type A strains did. Third, the XPR6 disruption strains grew poorly on rich media at pH 5.5 and above. Cells remained physically attached after budding and continued to bud forming large dog balloon-like structures. In addition, these structures aggregated forming visible clumps in liquid culture. These growth aberrations were largely eliminated by growing cells in medium at pH 4. Fourth, no mycelial forms were observed regardless of the pH.
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PMID:Cloning, nucleotide sequence and functions of XPR6, which codes for a dibasic processing endoprotease from the yeast Yarrowia lipolytica. 820 53

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

Cadmium is a potent poison for living cells. In man, chronic exposure to low levels of cadmium results in damage to kidneys and has been linked to neoplastic disease and ageing, and acute exposure can cause damage to a variety of organs and tissues. Cadmium reacts with thiol groups and can substitute for zinc in certain proteins, but the reason for its toxicity in vivo remains uncertain. In eukaryotes, an important selective proteolysis pathway for the elimination of abnormal proteins that are generated under normal or stress conditions is ATP-dependent and mediated by the ubiquitin system. Substrates of this pathway are first recognized by ubiquitin-conjugating enzymes (or auxiliary factors) which covalently attach ubiquitin, a small and highly conserved protein, to specific internal lysine residues of proteolytic substrates. Ubiquitinated substrates are then degraded by the proteasome, a multisubunit protease complex. Here we show that expression of this ubiquitin-dependent proteolysis pathway in yeast is activated in response to cadmium exposure and that mutants deficient in specific ubiquitin-conjugating enzymes are hypersensitive to cadmium. Moreover, mutants in the proteasome are hypersensitive to cadmium, suggesting that cadmium resistance is mediated in part by degradation of abnormal proteins. This indicates that a major reason for cadmium toxicity may be cadmium-induced formation of abnormal proteins.
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PMID:Resistance to cadmium mediated by ubiquitin-dependent proteolysis. 838 Dec 13

Binding of alpha factor to Ste2p, a G protein-coupled plasma membrane receptor, activates a signal transduction pathway and stimulates endocytosis of the receptor-ligand complex. Ligand binding also induces ubiquitination of the Ste2p cytoplasmic tail. Protein ubiquitination is required for stimulated endocytosis of Ste2p, as internalization is 5- to 15-fold slower in ubc mutants that lack multiple ubiquitin-conjugating enzymes. In a C-terminal truncated form of Ste2p that is rapidly ubiquitinated and endocytosed in response to ligand binding, a single lysine to arginine substitution in its cytoplasmic tail eliminates both ubiquitination and internalization. Thus, ubiquitination of Ste2p itself is required for ligand-stimulated endocytosis. We propose that ubiquitination mediates degradation of receptor-ligand complexes, not via the proteasome, but by acting as a signal for endocytosis leading to subsequent degradation in the lysosome/vacuole.
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PMID:Ubiquitination of a yeast plasma membrane receptor signals its ligand-stimulated endocytosis. 856 73

Ubiquitin conjugation is a signal for degradation of eukaryotic proteins by the 26S protease. Conjugation of a homopolymeric multiubiquitin chain to a substrate lysine residue results in 10-fold faster degradation than does conjugation of monoubiquitin, but the molecular basis of enhanced targeting by chains is unknown. We show that ubiquitin residues L8, I44, and V70 are critical for targeting. Mutation of pairs of these residues to alanine had little effect on attachment of ubiquitin to substrates but severely inhibited degradation of the resulting conjugates. The same mutations blocked the binding of chains to a specific subunit (S5a) of the regulatory complex of the 26S protease. The side chains implicated in this binding--L8, I44, and V70--form repeating patches on the chain surface. Thus, hydrophobic interactions between these patches and S5a apparently contribute to enhanced proteolytic targeting by multiubiquitin chains.
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PMID:Surface hydrophobic residues of multiubiquitin chains essential for proteolytic targeting. 857 Jun 49

Targeting of substrates for degradation by the ATP, ubiquitin-dependent pathway requires formation of multiubiquitin chains in which the 8.6-kDa polypeptide is linked by isopeptide bonds between carboxyl termini and Lys-48 residues of successive monomers. Binding of Lys-48-linked chains by subunit 5 of the 26 S proteasome regulatory complex commits the attached target protein to degradation with concomitant release of free ubiquitin monomers following disassembly of the chains. Point mutants of ubiquitin (Lys-->Arg) were used to map the linkage specificity for ubiquitin-conjugating enzymes previously demonstrated to form novel multiubiquitin chains not attached through Lys-48. Recombinant human E2EPF catalyzed multiubiquitin chain formation exclusively through Lys-11 of ubiquitin while recombinant yeast RAD6 formed chains linked only through Lys-6. Multiubiquitin chains linked through Lys-6, Lys-11, or Lys-48 each bound to subunit 5 of partially purified human 26 S proteasome with comparable affinities. Since chains bearing different linkages are expected to pack into distinct structures, competition between Lys-11 and Lys-48 chains for binding to subunit 5 demonstrates that the latter possesses determinants for recognizing alternatively linked chains and precludes the existence of subunit 5 isoforms recognizing distinct structures. In addition, competition studies provided an estimate of Kd < or = 18 nM for the intrinsic binding of Lys-48-linked chains of linkage number n > 4. This result suggests that the principal mechanistic advantage of multiubiquitin chain formation is to enhance the affinity of the associated substrate for the 26 S complex relative to that of unconjugated target protein. Complementation studies with E1/E2-depleted rabbit reticulocyte extract demonstrated RAD6 supported isopeptide ligase-dependent degradation only through Lys-48-linked chains, while E2EPF retained the ability to target a model radiolabeled substrate through Lys-11-linked chains. Therefore, the linkage specificity exhibited by these E2 isozymes depends on their catalytic context with respect to isopeptide ligase.
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PMID:Novel multiubiquitin chain linkages catalyzed by the conjugating enzymes E2EPF and RAD6 are recognized by 26 S proteasome subunit 5. 857 61

Endothelial cells play a major role in recruiting leukocytes to sites of inflammation. This is accomplished, at least in part, by up-regulation of cell surface adhesion molecules, including VCAM-1 and ICAM-1, in response to cytokines. In this report, we investigated the role of the proteasome complex in mediating the interleukin (IL)- 1 beta induction of VCAM-1 and ICAM-1 gene expression in human endothelial cells. We present evidence that a proteasome inhibitor, n-acetyl-leucinyl-leucinyl-norleucinal (norLEU), as well as specific protease inhibitors, n-tosyl-Lys-chloromethylketone and N-tosyl-Phe-chloromethylketone, blocked IL-1 beta induction of VCAM-1 and ICAM-1 promoter-driven reporter gene expression in stably transfected endothelial cells. These inhibitors also blocked cytokine induced cell surface expression of VCAM-1 and ICAM-1 by human umbilical vein endothelial cells. As expected, the protease inhibitors blocked the activation of nuclear factor (NF)-kappa B in response to IL-1 beta stimulation. In contrast, norLEU did not prevent IL-1 beta-induced nuclear translocation of NF-kappa B. The effects of norLEU were specific because it did not inhibit the IL-1 beta induction of plasminogen activator inhibitor type 1 gene expression. This study demonstrates that inhibition of the proteolytic activity of the proteasome blocks IL-1 beta induction of VCAM-1 and ICAM-1 gene expression in human endothelial cells.
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PMID:Proteasome inhibitors block VCAM-1 and ICAM-1 gene expression in endothelial cells without affecting nuclear translocation of nuclear factor-kappa B. 862 76

Covalent conjugation of ubiquitin to intracellular proteins is a signal for degradation by the 26S protease. Conjugation is usually accomplished by the sequential action of activating (E1), conjugating (E2), and ligase (E3) enzymes. Each of these enzymes forms a covalent thiol ester with ubiquitin as part of its catalytic cycle. In most cases, the apparent role of the ubiquitin conjugating enzyme (E2) is to transfer ubiquitin from the E1 active site to the E3 active site. Ubiquitin is then delivered from E3 to the substrate lysine residue. An unusually large, reticulocyte-specific enzyme, known as E2-230K, is unique among the large family of E2 enzymes is being susceptible to inhibition by inorganic arsenite [Klemperer et al. (1989) Biochemistry 28, 6035-6041]. We show that phenylarsenoxides potently inhibit E2-230K, apparently by binding to vicinal Cys residues of the enzyme: bound aminophenylarsenoxide partially protects the enzyme against inactivation by N-ethylmalemide (NEM), and prior enzyme inactivation with NEM blocks enzyme binding to immobilized phenylarsenoxide. Studies on the mechanistic basis of inhibition showed that a concentration of (aminophenyl)arsenoxide that produced complete inhibition of steady-state turnover had no effect on the turnover of the preformed E2-ubiquitin adduct. However, when the enzyme was preincubated with this concentration of inhibitor prior to initiation of adduct formation, the level of E2-associated ubiquitin was reduced by 60%. These results are consistent with a model in which two Cys residues of the enzyme sequentially form thiol esters with ubiquitin and the second of these Cys residues is bound to arsenic in the enzyme-inhibitor complex. In this model, E2-230K functions as an E2-E3 hybrid.
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PMID:Mechanism of ubiquitin conjugating enzyme E2-230K: catalysis involving a thiol relay? 863 98

We have previously shown that incubation of the model protein glucose-6-phosphate dehydrogenase (Glu-6-PDH) from the bacterium Leuconostoc mesenteroides with 4-hydroxy-2-nonenal (HNE), a major product of lipid peroxidation, results in the formation of cross-linked protein. HNE-modified protein is resistant to proteolytic degradation and acts as an inhibitor of the multicatalytic proteinase. It was therefore important to establish the chemistry of the cross-linking reaction. The formation of cross-linked Glu-6-PDH is associated with the nearly exclusive loss of lysine residues. For this reason the reaction of N-acetyllysine with HNE has been investigated. The epsilon-amino group of lysine reacts with the double bond (C3) and the carbonyl (C1) functions of HNE via Michael addition and Schiff base formation resulting in the production of a 2:1 amino acid-HNE cross-link. Chromatographic detection of this adduct in the acid hydrolysate of HNE-treated Glu-6-PDH reveals that this chemistry is responsible for the formation of cross-linked protein. Antibody to the reduced form of the 2:1 lysine-HNE adduct was prepared. The antibody was used to demonstrate that exposure of isolated liver mitochondria to oxidative stress led to the formation of intra- and intermolecular protein-HNE cross-links. The results of the present study indicate that modifications to protein by lipid peroxidation products may be physiologically relevant and could contribute to the disease- and age-related buildup of damaged protein.
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PMID:Chemical characterization of a protein-4-hydroxy-2-nonenal cross-link: immunochemical detection in mitochondria exposed to oxidative stress. 863 25

Activation of transcription factor NF-kappaB involves signal-induced degradation of the protein inhibitor IkappaB-alpha and release of NF-kappaB which translocates to the nucleus where it influences transcription of responsive genes. Although multiple regions of IkappaB-alpha are involved in this process, the N-terminal region of the protein has been identified as a regulatory region that is required for signal induced phosphorylation and degradation. The sensitivity of IkappaB-alpha degradation to peptide aldehydes which inhibit components of the proteasome and the detection of ubiquitinated forms of IkappaB-alpha indicate that IkappaB-alpha is degraded by the ubiquitin-proteasome pathway. To identify lysine residues that represent the sites of ubiquitin addition, a series of lysine to arginine mutations were introduced into IkappaB-alpha and the mutant proteins tested for their ability to function in vivo. Exposure of COS7 cells, cotransfected with IkappaB-alpha and a TNF-responsive NF-kappaB reporter gene, resulted in stimulation of reporter activity as a consequence of IkappaB-alpha degradation. In contrast, this effect was drastically reduced when an IkappaKB-alpha mutant carrying serine to alanine changes at amino-acids, 32 and 36, which blocks both signal-induced phosphorylation and ubiquitin conjugation of the protein, was co-transfected with the reporter gene. Likewise, a mutant form of IkappaB-alpha containing lysine to arginine changes at positions 21 and 22 (K21R, K22R) severely reduces TNF-induced activation of the NF-kappaB-dependent reporter gene. Examination of the metabolism of mutant IkappaB-alpha molecules reveals that, while the K21R, K22R mutant inhibits the DNA-binding activity of NF-kappaB and undergoes signal induced phosphorylation, it is neither ubiquitinated nor degraded in response to TNF. Thus, it is likely that after signal-induced phosphorylation Of IkappaB-alpha on serine residues 32 and 36, lysine residues 21 and 22 are major sites of ubiquitin ligation which target the protein for rapid degradation by the proteasome.
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PMID:Identification of lysine residues required for signal-induced ubiquitination and degradation of I kappa B-alpha in vivo. 864 84


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