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:3.2.1.17 (lysozyme)
21,489 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

In the ubiquitin (Ub) system for protein degradation, proteins ligated to Ub are degraded by an ATP-dependent 26 S protease complex. During or after proteolysis, free Ub is regenerated, but the mechanisms of Ub release remained unknown. It was previously observed that free Ub is released from a Ub-histone conjugate by an ATP-dependent activity present in partially purified preparations of 26 S complex, but the relationship of this activity to protein breakdown was not established. We now show that purified preparations of 26 S complex release free Ub from conjugates that are good substrates for proteolysis, such as conjugates of lysozyme with reductively methylated Ub. The activity that releases free Ub co-migrates with the 26 S protease complex in glycerol density gradient centrifugation, indicating that the responsible Ub C-terminal hydrolase is an integral part of the 26 S complex. Complex-associated hydrolase can also act on adducts in which a single Ub unit is attached to protein, such as a bacterially expressed construct in which the C terminus of Ub is fused to the alpha-NH2 group of a fragment of Ub that contains 60% of its N-terminal region. In all cases, Ub release is insensitive to Ub-aldehyde (an inhibitor of some Ub C-terminal hydrolases) and is stimulated by MgATP. ATP cannot be replaced by beta, gamma-nonhydrolyzable analogs, but it can be substituted by CTP and GTP. The nucleotide specificity of Ub release by the 26 S complex is similar to that observed previously for conjugate proteolysis and nucleotide hydrolysis. It thus seems that the activity of the Ub C-terminal hydrolase associated with the 26 S complex is tightly coupled to the proteolytic action of the complex, and it may have a role in the release of Ub from linkage to amino groups of the protein substrate at the final stages of the Ub proteolytic pathway.
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PMID:Ubiquitin C-terminal hydrolase activity associated with the 26 S protease complex. 838 22

Covalent attachment of ubiquitin marks substrates for proteolysis, but features that identify ubiquitination targets such as chicken egg white lysozyme are poorly understood. Recognition of lysozyme first requires reduction of Cys-6 Cys-127, one of its four native disulfide bonds, and Cys-6,Cys-127-carboxymethylated (6,127-rcm) lysozyme can mimic this three-disulfide intermediate. The 6,127-rcm form of lysozyme is known to retain a substantially native-like conformation in solution, and we demonstrate that it is this folded structure that is recognized for ubiquitination. Because native lysozyme is not a substrate, differences between the native and three-disulfide structures must include features responsible for selective ubiquitination. The 1.9-A resolution crystal structure of 6,127-rcm-lysozyme, reported here, affords a view of this ubiquitin-dependent degradation substrate. Two conformers of 6,127-rcm-lysozyme were obtained in the crystal. These differ uniquely from crystal forms of native lysozyme by displacement of the C-terminal residues. The structures suggest that localized unfolding at the C terminus of three-disulfide lysozyme allows the complex of E3 alpha (ubiquitin-protein ligase) and E2 (ubiquitin-carrier protein) to bind to a surface that includes Lys-1 and the putative ubiquitination site Lys-13. From this we infer that the N-terminal and internal substrate recognition sites on the E3 alpha.E2 complex are separated by approximately 20 A.
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PMID:Crystal structure of a ubiquitin-dependent degradation substrate: a three-disulfide form of lysozyme. 838 11

We examined ubiquitination and ubiquitin-mediated degradation of glycated protein by rabbit reticulocyte lysate fraction II (ubiquitin-free preparation). Non-glycated lysozyme and three glycated lysozyme preparations with different glucose binding ratios were used as substrates. Glycation sites of the lysozyme were mostly the epsilon-NH2 group of lysine residues, since modification at the alpha-NH2 group of the amino terminal was not detectable. Ubiquitin was conjugated with three glycated lysozyme preparations, which contained 1.4, 2.8 and 4.5 mol glucose per mol, by fraction II supplemented with hemin. The extent of formed conjugates was reduced 81, 72 and 56% of those of ubiquitin-non-glycated lysozyme conjugates, respectively. Additionally, ubiquitin-mediated degradation of the resultant conjugates was reduced and their respective rates were 97, 56 and 19% of that of the non-glycated lysozyme. These results indicated that both ubiquitin conjugation and ubiquitin-mediated degradation of the lysozyme were inhibited by nonenzymatic glucose binding to the lysozyme.
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PMID:Inhibitory effect of nonenzymatic glycation on ubiquitination and ubiquitin-mediated degradation of lysozyme. 838 87

Insoluble ubiquitin conjugates (UC) in the mitochondrial fraction of the gerbil cortex were analyzed following transient forebrain ischemia. At 1 h of reperfusion after 2-10 min of ischemia, UC increased as the duration of ischemia was prolonged. Pre-treatment with pentobarbital, rather than post-treatment immediately after recirculation, reduced the increase of UC at 1 h of reperfusion following 5 min of ischemia. Pentobarbital had no effect on in vitro ubiquitination of heat-denatured lysozyme by the extract of gerbil cortex. These results suggest that increase in UC is dependent on ischemic damage and pentobarbital attenuates the increase of UC by relieving injury during ischemia.
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PMID:Increase in ubiquitin conjugates dependent on ischemic damage. 840 94

Xenopus egg extract is capable of supporting mitosis in vitro, which makes it ideal for biochemical analysis of the cell cycle. Since several studies have implicated the ubiquitin system in cell cycle progression, we have measured ubiquitin conjugation rates, proteolysis of ubiquitin-lysozyme conjugates, and rates of isopeptidase activity in cycling Xenopus egg extracts. Although ubiquitin conjugation in cytostatic factor arrested extract was half that in activated extract, there were no changes in rates of ubiquitin conjugation during the cell cycle. Ubiquitin conjugates are degraded by a 26 S ATP-stimulated protease. The ability of the 26 S protease to degrade ubiquitin-lysozyme conjugates and a fluorigenic peptide also remained constant across the cell cycle. In contrast to previously characterized systems, isopeptidase activity in Xenopus egg extract is energy-dependent. Glycerol gradient fractionation of Xenopus egg extract separated two ATP-dependent isopeptidases. On co-sedimented with the 26 S protease; the other sedimented slower and was not associated with any additional proteolytic activity. As found for rates of Ub conjugation and conjugate proteolysis, there was little or no variation in isopeptidase activity during the cell cycle.
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PMID:Ubiquitin metabolism in cycling Xenopus egg extracts. 840 56

A variety of protease inhibitors have been used to study ubiquitin-dependent proteolysis by the 26 S protease. However, these inhibitors lack complete specificity and thus affect ubiquitin-independent pathways as well. We recently identified an Arabidopsis protein, MBP1, that is homologous to subunit 5a (S5a) of the human 26 S protease complex. MBP1 and S5a bind multiubiquitin chains with high affinity and presumably facilitate the recognition of ubiquitin conjugates by the 26 S protease. We show here that free MBP1 can be a potent inhibitor of ubiquitin-dependent proteolysis in several cell-free systems. When added to reticulocyte lysates or to Xenopus egg extracts, the plant protein effectively blocked the degradation of multiubiquitinated lysozyme and cyclin B, respectively. MBP1 did not enhance the removal of ubiquitin from lysozyme or affect the ability of the 26 S complex to hydrolyze fluorogenic peptides. These data suggest that the plant protein specifically interferes with the recognition of ubiquitin conjugates by the 26 S protease. Thus MBP1, human S5a, and their homologs should prove to be valuable reagents for investigating cellular events mediated by ubiquitin-dependent proteolysis.
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PMID:Inhibition of ubiquitin-mediated proteolysis by the Arabidopsis 26 S protease subunit S5a. 853 Mar 51

A ubiquitin (Ub)/ATP-dependent proteolytic complex (26S proteasome) purified from rabbit skeletal muscle was dissociated into two subcomplexes, a 20S proteasome and a regulatory subunit complex, by preparative non-denaturing polyacrylamide gel electrophoresis (PAGE). The isolated regulatory subunit complex preparation gave a single broad band on analytical non-denaturing PAGE, and several bands ranging between 33 and 110 kDa on SDS-PAGE. This complex was found to consist of about 20 subunits on the basis of two-dimensional PAGE, the pattern of which appeared identical or very similar to that of the 33-110 kDa 26S proteasome subunits. The apparent molecular mass of the complex was estimated to be 1100 kDa by Ferguson plot analysis and also by Superose 6 gel filtration. Unlike the 26S proteasome, neither ATPase activity nor protease activities toward Suc-Leu-Leu-Val-Tyr-MCA, Boc-Phe-Ser-Arg-MCA, Z-Leu-Leu-Glu-beta NA, [14C]-casein, [125I]-lysozyme and Ub-[125I]-lysozyme were significantly detectable in the regulatory subunit complex. This complex was found to be capable of associating with itself in MgATP-dependent manner. These results suggest that a regulatory subunit complex dissociated from the 26S proteasome comprises all the higher molecular mass subunits of the 26S proteasome, and has no detectable ATPase and protease activities, although the homo-oligomerization occurs in an ATP-dependent fashion.
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PMID:Regulatory subunit complex dissociated from 26S proteasome: isolation and characterization. 854 9

Degradation of a protein via the ubiquitin system involves two discrete steps, conjugation of ubiquitin to the substrate and degradation of the adduct. Conjugation follows a three-step mechanism. First, ubiquitin is activated by the ubiquitin-activating enzyme, E1. Following activation, one of several E2 enzymes (ubiquitin-carrier proteins or ubiquitin-conjugating enzymes, UBCs) transfers ubiquitin from E1 to the protein substrate that is bound to one of several ubiquitin-protein ligases, E3s. These enzymes catalyze the last step in the process, covalent attachment of ubiquitin to the protein substrate. The binding of the substrate to E3 is specific and implies that E3s play a major role in recognition and selection of proteins for conjugation and subsequent degradation. So far, only a few ligases have been identified, and it is clear that many more have not been discovered yet. Here, we describe a novel ligase that is involved in the conjugation and degradation of non "N-end rule" protein substrates such as actin, troponin T, and MyoD. This substrate specificity suggests that the enzyme may be involved in degradation of muscle proteins. The ligase acts in concert with E2-F1, a previously described non N-end rule UBC. Interestingly, it is also involved in targeting lysozyme, a bona fide N-end substrate that is recognized by E3 alpha and E2-14 kDa. The novel ligase recognizes lysozyme via a signal(s) that is distinct from the N-terminal residue of the protein. Thus, it appears that certain proteins can be targeted via multiple recognition motifs and distinct pairs of conjugating enzymes. We have purified the ligase approximately 200-fold and demonstrated that it is different from other known E3s, including E3 alpha/UBR1, E3 beta, and E6-AP. The native enzyme has an apparent molecular mass of approximately 550 kDa and appears to be a homodimer. Because of its unusual size, we designated this novel ligase E3L (large). E3L contains an -SH group that is essential for its activity. Like several recently described E3 enzymes, including E6-AP and the ligase involved in the processing of p105, the NF-kappa B precursor, the novel ligase is found in mammalian tissues but not in wheat germ.
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PMID:Isolation, characterization, and partial purification of a novel ubiquitin-protein ligase, E3. Targeting of protein substrates via multiple and distinct recognition signals and conjugating enzymes. 855 May 77

ATP-dependent proteolysis of 125I-labeled human alpha-globin, bovine alpha-lactalbumin, bovine serum albumin, or chicken lysozyme was assessed in a rabbit reticulocyte extract supplemented with ATP, excess ubiquitin, and variable amounts of ubiquitin aldehyde (Ubal), an inhibitor of many ubiquitin-protein isopeptidases. Low concentrations (0.8 microM) of Ubal increased the ATP-dependent degradation of 125I-alpha-globin by approximately 30% after 2 h at 37 degrees C, had little effect on 125I-lysozyme turnover, and decreased 125I-alpha-lactalbumin or 125I-albumin degradation by approximately 20%. The ATP-dependent degradation of all substrates was inhibited by high concentrations (> 3 microM) of Ubal throughout the incubation (15 min to 2 h); after 2 h, this inhibition ranged from 15% for 125I-alpha-globin to approximately 85% for 125I-alpha-lactalbumin and 125I-albumin. Levels of ubiquitin-125I-protein conjugates were increased significantly with Ubal; with > or = 8.0 microM Ubal, high molecular mass multiubiquitinated conjugates were particularly evident for 125I-alpha-globin and 125I-alpha-lactalbumin. These mixtures also accumulated ubiquitin conjugates with sizes expected for di- through pentaubiquitin oligomers. The results are consistent with the following proposed events: The ATP-dependent degradation of 125I-alpha-lactalbumin or 125I-albumin is probably mediated almost exclusively through polyubiquitinated intermediates. High Ubal concentrations inhibit an isopeptidase(s) which normally disassembles "unanchored" polyubiquitin chains that remain after substrate degradation by the 26S proteasome; these chains accumulate to inhibit further conjugate degradation. Much of the ATP-dependent degradation of 125I-alpha-globin and, to a lesser degree, 125I-lysozyme may occur through alternative structures where ubiquitin monomers or short oligomers are ligated to one or more substrate lysines. For 125I-alpha-globin, even low concentrations of Ubal effectively inhibit deubiquitination of these conjugates to enhance alpha-globin degradation.
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PMID:Differential effects of ubiquitin aldehyde on ubiquitin and ATP-dependent protein degradation. 871 81

Free ubiquitin (mainly monoubiquitin) and multi-ubiquitin chains coexist in eukaryote cells and serve distinct cellular roles. However, any immunoassay systems established previously have not been proved to be applicable for measuring the former without cross-reactive responses with the latter. For this purpose, we developed a radioimmunoassay specific to monoubiquitin by employing antiserum US-1 against ubiquitin. In this assay, ubiquitin-protein conjugates, prepared by a reticulocyte lysate fraction II and fractionated on Moro Q and Superdex 200 columns, exhibited practically no cross-reactivity. The cross-reactivity of fractionated ubiquitin-lysozyme conjugates was also analyzed as a function of their multi-ubiquitin chain size. As a result, the larger the conjugates were found to be, the weaker were the cross-reactive responses they showed, and the multi-ubiquitin chains (n > approx. 20) were substantially unreactive in the radioimmunoassay. By using the radioimmunoassay, heat-shock-induced decrease in the level of cellular free (mono)ubiquitin was detected. In addition, the standard preparation of multi-ubiquitin chains was not cross-reactive in all other five radioimmunoassays employing distinct antibodies to ubiquitin (four antisera and a monoclonal antibody). These data suggest that radioimmunoassays employing ubiquitin antibodies raised by the general methods can discriminate between monoubiquitin and multi-ubiquitin chains and quantitate cellular free ubiquitin.
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PMID:Ability of ubiquitin radioimmunoassay to discriminate between monoubiquitin and multi-ubiquitin chains. 876 32


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