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

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

Recent studies have suggested that activation of the ubiquitin-proteasome pathway is primarily responsible for the rapid loss of muscle proteins in various types of atrophy. The present studies were undertaken to test if different classes of muscle proteins are degraded by this pathway. In extracts of rabbit psoas muscle, the complete degradation of soluble proteins to amino acids was stimulated up to 6-fold by ATP. Peptide aldehyde inhibitors of the proteasome or the removal of proteasomes markedly inhibited only the ATP-dependent process. Addition of purified myosin, actin, troponin, or tropomyosin to these extracts showed that these proteins served as substrates for the ubiquitin-proteasome pathway. By contrast, degradation of myoglobin did not require ATP, proteasomes, or any known proteases in muscles. When myosin, actin, and troponin were added as actomyosin complexes or as intact myofibrils to these extracts, they were not hydrolyzed at a significant rate, probably because in these multicomponent complexes, these proteins are protected from degradation. Accordingly, actin (but not albumin or troponin) inhibited the degradation of 125I-myosin, and actin was found to selectively inhibit ubiquitin conjugation to 125I-myosin. Also, the presence of tropomyosin inhibited the degradation of 125I-troponin. However, neither actin nor tropomyosin inhibited the degradation of 125I-lysozyme or soluble muscle proteins. Thus, specific interactions between the myofibrillar proteins appear to protect them from ubiquitin-dependent degradation, and the rate-limiting step in their degradation is probably their dissociation from the myofibril.
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PMID:Importance of the ATP-ubiquitin-proteasome pathway in the degradation of soluble and myofibrillar proteins in rabbit muscle extracts. 890 Jan 46

A crude fraction that contains ubiquitin-protein ligases contains also a proteolytic activity of approximately 100 kDa that cleaves p53 to several fragments. The protease does not require ATP and is inhibited in the crude extract by an endogenous approximately 250 kDa inhibitor. The proteinase can be inhibited by chelating the Ca2+ ions, by specific cysteine proteinase inhibitors and by peptide aldehyde derivatives that inhibit calpains. Purified calpain demonstrates an identical activity that can be inhibited by calpastatin, the specific protein inhibitor of the enzyme. Thus, it appears that the activity we have identified in the extract is catalyzed by calpain. The calpain in the extract degrades also N-myc, c-Fos and c-Jun, but not lysozyme. In crude extract, the calpain activity can be demonstrated only when the molar ratio of the calpain exceeds that of its native inhibitor. Recent experimental evidence implicates both the ubiquitin proteasome pathway and calpain in the degradation of the tumor suppressor, and it was proposed that the two pathways may play a role in targeting the protein under various conditions. The potential role of the two systems in this important metabolic process is discussed.
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PMID:On the involvement of calpains in the degradation of the tumor suppressor protein p53. 910 77

A cDNA encoding a new ubiquitin-specific protease, UBP41, in chick skeletal muscle was cloned using an Escherichia coli-based in vivo screening method. Nucleotide sequence analysis of the cDNA containing an open reading frame of 1,071 base pairs revealed that the protease consists of 357 residues with a calculated molecular mass of 40,847 Da, and is related to members of the UBP family containing highly conserved Cys and His domains. Chick UBP41 was expressed in E. coli and purified from the cells to apparent homogeneity, using 125I-labeled ubiquitin-alphaNH-MHISPPEPESEEEEEHYC as a substrate. The purified enzyme behaved as an approximately 43-kDa protein under both denaturing and nondenaturing conditions, suggesting that it consists of a single polypeptide chain. Like other deubiquitinating enzymes, it was sensitive to inhibition by ubiquitin-aldehyde and sulfhydryl blocking agents, such as N-ethylmaleimide. The UBP41 protease cleaved at the C terminus of the ubiquitin moiety in natural and engineered fusions irrespective of their sizes; thus, it is active against ubiquitin-beta-galactosidase as well as ubiquitin C-terminal extension protein of 80 amino acids. UBP41 also released free ubiquitin from poly-His-tagged di-ubiquitin. Moreover, it converted poly-ubiquitinated lysozyme conjugates to mono-ubiquitinated forms of about 24 kDa, although the latter molecules were not further degraded to free ubiquitin and lysozyme. These results suggest that UBP41 may play an important role in the recycling of ubiquitin by hydrolysis of branched poly-ubiquitin chains generated by the action of 26 S proteasome on poly-ubiquitinated protein substrates, as well as in the production of free ubiquitin from linear poly-ubiquitin chains and of certain ribosomal proteins from ubiquitin fusion proteins.
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PMID:Molecular cloning of a novel ubiquitin-specific protease, UBP41, with isopeptidase activity in chick skeletal muscle. 932 73

Cell-permeant peptidyl aldehydes and diazomethylketones are frequently utilized as inhibitors of regulatory intracellular proteases. In the present study the specificities of several peptidyl inhibitors for purified human mu-calpain and 20 S proteasome were investigated. Acetyl-LLnL aldehyde, acetyl-LLM aldehyde, carbobenzyloxy-LLnV aldehyde (ZLLnVal), and carbobenzyloxy-LLY-diazomethyl ketone produced half-maximum inhibition of the caseinolytic activity of mu-calpain at concentrations of 1-5 x 10(-7) M. In contrast, only ZLLnVal was a reasonably potent inhibitor of the caseinolytic activity of 20 S proteasome, producing 50% inhibition at 10(-5) M. The other inhibitors were at least 10-fold less potent, producing substantial inhibition only at near saturating concentrations in the assay buffer. Further studies with ZLLnVal demonstrated that its inhibition of the proteasome was independent of casein concentration over a 25-fold range. Proteolysis of calpastatin or lysozyme by the proteasome was half-maximally inhibited by 4 and 22 microM ZLLnVal, respectively. Thus, while other studies have shown that ZLLnVal is a potent inhibitor of the hydrophobic peptidase activity of the proteasome, it appears to be a much weaker inhibitor of its proteinase activity. The ability of the cell permeant peptidyl inhibitors to inhibit growth of the yeast Saccharomyces cerevisiae was studied because this organism expresses proteasome but not calpains. Concentrations of ZLLnVal as high as 200 microM had no detectable effect on growth rates of overnight cultures. However, yeast cell lysates prepared from these cultures contained 2 microM ZLLnVal, an amount which should have been sufficient to fully inhibit hydrophobic peptidase activity of yeast proteasome. Degradation of ubiquitinylated proteins in yeast extracts by endogenous proteasome was likewise sensitive only to high concentrations of ZLLnVal. The higher sensitivity of the proteinase activity of calpains to inhibition by the cell permeant inhibitors suggests that calpain-like activities may be targets of these inhibitors in animal cells.
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PMID:Specificities of cell permeant peptidyl inhibitors for the proteinase activities of mu-calpain and the 20 S proteasome. 936 65

Covalent linkage of poly(ethylene glycol) (PEG) to drug molecules results in water-soluble conjugates with altered bioavailability, pharmacokinetics, immunogenic properties, and biological activities. For drugs bearing one or more amino groups, reductive amination is a potentially useful method for conjugation to PEG. PEG acetaldehyde has been used for this purpose, but its ease of polymerization under certain conditions and its susceptibility to air oxidation have caused some problems in its application. A simple and reliable method for preparation and use in reductive amination of PEG acetaldehyde hydrate generated in situ by hydrolysis of PEG acetaldehyde diethylacetal is demonstrated. PEG acetaldehyde diethylacetal is prepared in high yield and purity by reaction of PEG with chlorodiethylacetal in dioxane in the presence of finely powdered sodium hydroxide under heterogeneous conditions. PEG acetaldehyde hydrate is generated in solution by hydrolysis in aqueous acids. Solutions of the hydrate may be used directly, in conjunction with sodium cyanoborohydride, to effect reductive amination. We demonstrate application of these methods in PEGylation of lysozyme and chitosan to form water-soluble methoxy poly(ethylene glycol) (mPEG) derivatives and PEG-chitosan hydrogels.
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PMID:Reductive amination using poly(ethylene glycol) acetaldehyde hydrate generated in situ: applications to chitosan and lysozyme. 981 4

The process of degranulation of mast cells and neutrophils contributes to inflammatory disorders. Activation of microglial cells and macrophages is believed to be involved in inflammatory, infectious and degenerative diseases of the CNS. Combining the potent inhibition of chemical mediators released by the degranulation of mast cells or neutrophils and from the activated microglial cells or macrophages, would lead to a promising anti-inflammatory agent for the treatment of peripheral and central inflammation. A series of chalcone derivatives have been reported to have potent anti-inflammatory activity. In an effort to continually develop potent anti-inflammatory agents, novel series of chalcones, 2'-hydroxy- and 2',5'-dihydroxychalcones were synthesized and their inhibitory effects on the activation of mast cells, neutrophils, microglial cells and macrophages were evaluated in-vitro. The chalcones were prepared by Claisen-Schmidt condensation of appropriate acetophenones with an appropriate aromatic aldehyde. The alkoxychalcones were prepared with appropriate hydroxychalcones and alkyl iodide and the dihydroxychalcones were prepared by hydrogenation of an appropriate chalcone with Pd/C. Almost all of the hydroxychalcones exhibited potent inhibitory effects on the release of beta-glucuronidase and lysozyme from rat neutrophils stimulated with formyl-Met-Leu-Phe/cytochalasin B (fMLP/CB). Of the hydroxychalcones, compound 1 was the most potent inhibitor of the release of beta-glucuronidase (IC50=1.6+/-0.2 microM) and lysozyme (IC50=1.4+/-0.2 microM) from rat neutrophils stimulated with fMLP/CB. Almost all of the 2',5'-dialkoxychalcones exhibited potent inhibitory effects on nitric oxide (NO) formation from murine microglial cell lines N9 stimulated with lipopolysaccharide (LPS). Of these, compound 11 showed the greatest effect (IC50=0.7+/-0.06 microM). The present results demonstrated that most of the chalcone derivatives have an anti-inflammatory effect. The inhibitory effects of dialkoxychalcones, 10-12 on inflammation are probably not due to the inhibition of mast cells and neutrophil degranulation, but are mediated through the suppression of NO formation from N9 cells.
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PMID:Synthesis and anti-inflammatory effect of chalcones. 1071 46

The immobilization of protein molecules on self-assembled monolayers (SAM) by physical interactions and chemical bonding has been studied using atomic force microscopy (AFM). The proteins used for our investigation are bovine serum albumin (BSA), lysozyme (LYZ), and normal rabbit immunoglobulin G (IgG). The surfaces are methyl-, hydroxyl-, carboxylic acid- and aldehyde-terminated SAMs. We found that BSA and LYZ can be readily immobilized on SAMs at their isoelectric point (IEP). The detailed surface morphology of adsorbed proteins varies with the functionality of the SAMs. The strong hydrophobic interaction at the IEP is attributed to immobilization. If the solution pH is deviated from the IEP, proteins may be attached onto the surface via electrostatic interactions. Covalent binding between the aldehyde-terminated SAM and the H2N-groups in the protein results in immobilization of all three proteins. The individual proteins and their orientations on SAMs are clearly resolved from high-resolution AFM images. The stability and bioactivity of these immobilized proteins are also studied.
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PMID:Immobilization of proteins on self-assembled monolayers. 1114 64

The increasing development of bacterial resistance to traditional antibiotics has reached alarming levels, thus necessitating the strong need to develop new antimicrobial agents. These new antimicrobials should possess both novel modes of action as well as different cellular targets compared with the existing antibiotics. Lysozyme, muramidase, and aprotinin, a protease inhibitor, both exhibit antimicrobial activities against different microorganisms, were chosen as model proteins to develop more potent bactericidal agents with broader antimicrobial specificity. The antibacterial specificity of lysozyme is basically directed against certain Gram-positive bacteria and to a lesser extent against Gram-negative ones, thus its potential use as antimicrobial agent in food and drug systems is hampered. Several strategies were attempted to convert lysozyme to be active in killing Gram-negative bacteria which would be an important contribution for modern biotechnology and medicine. Three strategies were adopted in which membrane-binding hydrophobic domains were introduced to the catalytic function of lysozyme, to enable it to damage the bacterial membrane functions. These successful strategies were based on either equipping the enzyme with a hydrophobic carrier to enable it to penetrate and disrupt the bacterial membrane, or coupling lysozyme with a safe phenolic aldehyde having lethal activity toward bacterial membrane. In a different approach, proteolytically tailored lysozyme and aprotinin have been designed on the basis of modifying the derived peptides to confer the most favorable bactericidal potency and cellular specificity. The results obtained from these strategies show that proteins can be tailored and modelled to achieve particular functions. These approaches introduced, for the first time, a new conceptual utilization of lysozyme and aprotinin, and thus heralded a great opportunity for potential use in drug systems as new antimicrobial agent.
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PMID:Strategies for new antimicrobial proteins and peptides: lysozyme and aprotinin as model molecules. 1194 64


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