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)

Fas (APO1/CD95) is a type 1 transmembrane protein critically involved in receptor-mediated apoptosis. Previous studies have shown that Fas exists in monomeric form in resting cells and aggregates upon cross-linking to form a complex that serves to recruit additional signaling molecules to the cell membrane. To study the molecular fate of the Fas antigen following receptor activation, a monoclonal antibody specific for the cell death domain of Fas has been generated. This monoclonal antibody (3D5) could be used in Western blot analysis using total cell lysates to identify different forms of Fas antigens without immunoprecipitation. High molecular mass (>200 kDa), SDS- and beta-mercaptoethanol-resistant Fas aggregates were formed immediately following receptor cross-linking, and a 97-kDa band (p97) was detected about 2 h later. p97 could be detected by antibodies against either the death domain or the C terminus. However, p97 could not be precipitated by antiextracellular domain antibodies. Thus, p97 most likely represents a processed form of the high molecular weight Fas aggregates. Although p97 generation followed a similar time course as CPP32 activation and poly(ADP-ribose) polymerase cleavage, it could not be inhibited by cysteine protease, calpain, or proteasome inhibitors.
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PMID:Activation-induced aggregation and processing of the human Fas antigen. Detection with cytoplasmic domain-specific antibodies. 926 81

Topoisomerase I (TOP1) relaxes superhelical DNA through a breakage/rejoining reaction in which the active site tyrosine links covalently to a 3' phosphate at the break site as a transient intermediate. The antitumor drug camptothecin (CPT) and its analogs inhibit the rejoining step of the breakage/rejoining reaction, which traps the enzyme in covalent linkage with DNA (the cleavable complex). Little is known about the fate of cellular TOP1 trapped in the cleavable complex. We have analyzed TOP1 in mammalian cell lines treated with CPT. When CPT-treated cells were lysed with either SDS or alkali and analyzed by Western blotting, greater than 90% of the TOP1 was linked to DNA. Nuclease treatment of the cell lysate to remove the covalently linked DNA from TOP1 revealed a distinct ladder of higher molecular weight bands having properties indicative of multi-ubiquitin (Ub) conjugates of TOP1. Approximately 5-10% of TOP1 was present as these conjugates within minutes of CPT treatment. Consistent with ubiquitination, TOP1 was not modified in ts85 cells at the restrictive temperature for its thermolabile ubiquitin-activating enzyme (E1). Because conjugation with ubiquitin can mark proteins for destruction by the 26S proteasome, we analyzed TOP1 protein levels during prolonged CPT treatment. TOP1 protein levels were reduced to about 25% during CPT treatments of 2-4 h resulting from increased destruction, with the half-life dropping from 10-16 h down to 1-2 h. The destruction of TOP1, like the formation of Ub-TOP1 conjugates, was not observed in ts85 cells at the restrictive temperature. The destruction of TOP1 was also prevented in cells treated with MG-132 and lactacystin, specific inhibitors of the 26S proteasome. Finally, the multi-Ub conjugates of TOP1 were observed whether or not aphidicolin was included in cotreatment with CPT, indicating that replication fork activity was not involved in making TOP1 a substrate for ubiquitination. These results demonstrate that independent of DNA replication, the TOP1 cleavable complex is ubiquitinated and destroyed in cells treated with antitumor drugs that block the religation step of the TOP1 reaction.
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PMID:Ubiquitin-dependent destruction of topoisomerase I is stimulated by the antitumor drug camptothecin. 930 65

20 and 26 S proteasomes were isolated from rat liver. The procedure developed for the 26 S proteasome resulted in greatly improved yields compared with previously published methods. A comparison of the kinetic properties of 20 and 26 S proteasomes showed significant differences in the kinetic characteristics with certain substrates and differences in the effects of a protein substrate on peptidase activity. Observed differences in the kinetics of peptidylglutamyl peptide hydrolase activity suggest that the 26 S complex cannot undergo the conformational changes of 20 S proteasomes at high concentrations of the substrate benzyloxycarbonyl (Z) -Leu-Leu-Glu-beta-naphthylamide. Various inhibitors that differentially affect the trypsin-like and chymotrypsin-like activities have been identified. Ala-Ala-Phe-chloromethyl (CH2Cl) inhibits chymotrypsin-like activity assayed with succinyl (Suc) -Leu-Leu-Val-Tyr-AMC, but surprisingly not hydrolysis of Ala-Ala-Phe-7-amido4-methylcoumarin (AMC). Tyr-Gly-Arg-CH2Cl inhibits Suc-Leu-Leu-Val-Tyr-AMC hydrolysis as well as trypsin-like activity measured with t-butoxycarbonyl (Boc) -Leu-Ser-Thr-Arg-AMC, while Z-Phe-Gly-Tyr-diazomethyl (CHN2) was found to inhibit only the two chymotrypsin-like activities. Radiolabeled forms of peptidyl chloromethane and peptidyl diazomethane inhibitors, [3H]acetyl-Ala-Ala-Phe-CH2Cl, [3H]acetyl- and radioiodinated Tyr-Gly-Arg-CH2Cl, and Z-Phe-Gly-Tyr-(125I-CHN2), have been used to identify catalytic components associated with each of the three peptidase activities. In each case, incorporation of the label could be blocked by prior treatment of the proteasomes with known active site-directed inhibitors, calpain inhibitor 1 or 3, 4-dichloroisocoumarin. Subunits of labeled proteasomes were separated either by reverse phase-HPLC and SDS-polyacrylamide gel electrophoresis or by two-dimensional polyacrylamide gel electrophoresis followed by autoradiography/fluorography and immunoblotting with subunit-specific antibodies. In each case, label was found to be incorporated into subunits C7, MB1, and LMP7 but in different relative amounts depending on the inhibitor used, consistent with the observed effects on the different peptidase activities. The results strongly suggest a relationship between trypsin-like activity and chymotrypsin-like activity. They also help to relate the different subunits of the complex to the assayed multicatalytic endopeptidase activities.
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PMID:Catalytic properties of 26 S and 20 S proteasomes and radiolabeling of MB1, LMP7, and C7 subunits associated with trypsin-like and chymotrypsin-like activities. 931 91

The proteasome (EC 3.4.99.46) is a high molecular mass (approximately 700 kDa) multisubunit enzyme complex which is the focus of worldwide research in order to identify the structure, mechanism of action and specificity of the complex. The purpose of the present study was to investigate the tryptic, chymotryptic and peptidylglutamyl-peptide hydrolysing (PGPH) activities of ostrich liver proteasome. The proteasome was purified from ostrich liver by employing ammonium sulphate fractionation, followed by three sequential chromatographic steps on Toyopearl Super Q-650 S, Sephadex G-150 and phenyl-Toyopearl columns. Temperature and pH optima were examined and the effect of inhibitors, detergents, fatty acids and cations on the peptidase activities was determined. Ostrich proteasome exhibited a relative M(r) of approximately 665,000 using non-denaturing gradient PAGE and dissociated into the characteristic "ladder" associated with the proteasome subunits during SDS-PAGE. The pH optima for the peptidase activities were found to be slightly alkaline (tryptic activity) and neutral (chymotryptic-like and PGPH activities). Ostrich liver proteasome was found to be activated in terms of the PGPH activity by fatty acids and SDS, whereas the chymotryptic and tryptic-like activities were differentially inhibited. Ostrich proteasome, in its inhibition by monovalent cations, was similar to the proteasomes extracted from other sources. The specificity of the proteasome appears to be very broad, although it lacks aminopeptidase activity. The yield compared favourably with similar extraction procedures which have been reported. On the basis of the physicochemical and kinetic properties which ostrich liver proteasome exhibited, it can be safely concluded that it corresponds well with the proteasomes isolated from many other sources.
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PMID:Purification and characterization of proteasome from ostrich liver. 936 39

Pili of Neisseria gonorrhoeae and Neisseria meningitidis mediate binding of the bacteria to human cell-surface receptors. We found that purified pili bound to a 55- to 60-kDa doublet band on SDS-PAGE of separated human epithelial cell extracts. This is a migration pattern typical of membrane cofactor protein (MCP or CD46). MCP is a widely distributed human complement regulatory protein. Attachment of the bacteria to epithelial cells was blocked by polyclonal and monoclonal antibodies directed against MCP, suggesting that this complement regulator is a receptor for piliated Neisseria. We proved this hypothesis by demonstrating that piliated, but not non-piliated, gonococci bound to CHO cells transfected with human MCP-cDNA. We also demonstrated a direct interaction between purified recombinant MCP and piliated Neisseria. Finally, recombinant MCP protein produced in E. coli inhibited attachment of the bacteria to target cells. Taken together, our data show that MCP is a human cell-surface receptor for piliated pathogenic Neisseria.
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PMID:Membrane cofactor protein (MCP or CD46) is a cellular pilus receptor for pathogenic Neisseria. 937 94

Human membrane cofactor protein (MCP, CD46) has been suggested, although no convincing evidence has been proposed, to be a fertilization-associated protein, in addition to its primary functions as a complement regulator and a measles virus receptor. We have cloned a cDNA encoding the murine homologue of MCP. This cDNA showed 45% identity in deduced protein sequence and 62% identity in nucleotide sequence with human MCP. Its ectodomains were four short consensus repeats and a serine/threonine-rich domain, and it appeared to be a type 1 membrane protein with a 23-amino acid transmembrane domain and a short cytoplasmic tail. The protein expressed on Chinese hamster ovary cell transfectants was 47 kDa on SDS/PAGE immunoblotting, approximately 6 kDa larger than the murine testis MCP. It served as a cofactor for factor I-mediated inactivation of the complement protein C3b in a homologous system and, to a lesser extent, in a human system. Strikingly, the major message of murine MCP was 1.5 kb and was expressed predominantly in the testis. It was not detected in mice defective in spermatogenesis or with immature germ cells (until 23 days old). Thus, murine MCP may be a sperm-dominant protein the message of which is expressed selectively in spermatids during germ-cell differentiation.
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PMID:Molecular cloning of a murine homologue of membrane cofactor protein (CD46): preferential expression in testicular germ cells. 946 5

A protease which was activated by SDS was purified to homogeneity from maize leaves. On the basis of its proteolytic activity towards ribulose-1,5-bisphosphate carboxylase/ oxygenase (Rubisco) or a synthesized peptide, the purification was carried out using immunoaffinity chromatography with a monoclonal antibody raised against a partially purified enzyme by native gradient PAGE. The purified protease showed three bands at 40, 15, and 13 kDa on SDS-PAGE, indicating that it was composed of heterogeneous subunits. The protease was specifically activated by SDS (optimum = 0.4% for Rubisco proteolysis), but not by poly-L-lysine, fatty acids, or ATP. The protease had a pH optimum around 4.9. beta-Mercaptoethanol stimulated the activity only in the presence of SDS. The proteolytic activity was sensitive to E-64 and leupeptin but was resistant to EDTA, suggesting that the enzyme was an SH-protease. Thus, this enzyme is a novel type of SDS-dependent protease which differs from proteasome, matrix metalloproteinase, and other proteases reported in many organisms.
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PMID:Purification of a novel type of SDS-dependent protease in maize using a monoclonal antibody. 951 7

The human monocyte chemoattractant protein-1 (MCP-1) was expressed at high levels in Pichia pastoris with the alcohol oxidase promoter. It was secreted from the yeast when either its natural signal sequence or the Saccharomyces cerevisiae alpha-factor signal peptide was used. SDS-PAGE and Western blot revealed two immunoreactive MCP-1 species at 15 and 8.5 kDa designated MCP-1H and MCP-1L, respectively; both were purified by cation-exchange chromatography. MCP-1H could be converted to MCP-1L by treatment with peptide N-glycosidase F, showing that the former is an N-glycosylated form of the latter. Laser desorption mass spectrometry showed that MCP-1L actually consisted of a mixture of three polypeptides of 8449, 8614, and 8780 Da and MCP-1H showed a broad peak at 11,134 Da. N-terminal peptide sequencing indicated that nearly half of MCP-1L lacked the two N-terminal amino acids found in the native protein. Both MCP-1H and MCP-1L could induce monocyte migration and calcium influx in THP-1 monocytic leukemia cells, although these activities were about 10- to 100-fold lower than those of MCP-1 produced in insect cells.
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PMID:Expression of human monocyte chemoattractant protein-1 in the yeast Pichia pastoris. 951 54

The proteasome is a cytoplasmic high-molecular-weight structure composed of several smaller protein and RNA subunits. It has been associated with non-lysosomal pathways of intracellular degradation, expressing multicatalytic proteinase activities and specific RNase activity. By standard methods, we have isolated andpartially purified proteasomes from human epidermis. We obtained the expected multiple 24-32 kDa subunits by SDS-PAGE, and evidence of RNA. Proteasomes degraded casein, as well as chromogens for t-PA and trypsin but not for chymotrypsin, these proteolytic activities overlap, but do not coincide with those observed in other organs. We found that human epidermal 28 S and 18 S rRNAs were degraded, but yeast RNA was not. By means of zymography, we demonstrated, for the first time, that RNase activity persists after dissociation of the proteasome on the gel and that it co-localizes to the same range of molecular weight subunits as the proteinase activity.
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PMID:Proteasomal RNase activity in human epidermis. 962 96

The lens of the human eye is a suitable model for age-related alterations at the molecular level. Age-related cataract formation is closely related to the accumulation of oxidatively altered proteins. In this study the influence of UV-A, UV-B, and UV-C irradiation on the proteolytic susceptibility of alpha-, betaL-, and betaH-crystallins by the isolated 20S proteasome was investigated. The proteins were irradiated with 280, 300, and 350 nm monochromatic light. Changes of the physical properties of the crystallins were characterized by absorbance measurements at 280 nm, fluorescence spectra, and SDS-PAGE-electrophoresis. The proteolytic susceptibility of crystallins was maximal after irradiation at 280 nm and three- to fivefold lower at 300 nm. Irradiation at 350 nm was not able to initiate proteolysis, probably due to protein-aggregate formation of higher molecular weight, as shown by SDS-PAGE. The damage of crystallins by UV-C light might be a signal for its proteolytic degradation by the 20S proteasome, whereas UV-B and UV-A do not increase the proteolytic susceptibility to the same extent but promote the formation of crosslinked proteins. Therefore, irradiation with UV, which is not followed by an increase in the proteolytic susceptibility, is accompanied by the formation of crosslinked proteins. It was concluded, that also long UV-B and UV-A may be involved in age-related alterations of the human lens and cataract formation.
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PMID:Dose- and wavelength-dependent oxidation of crystallins by UV light--selective recognition and degradation by the 20S proteasome. 964 Dec 54


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