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)

The nucleotide sequence of a cDNA that encodes a new subunit, named RC7-I, of the 20 S proteasome of rat hepatoma cells has been determined. The polypeptide predicted from the open reading frame consists of 201 amino acid residues with a calculated molecular weight of 22,912 and isoelectric point of 7.25. Approximately 80% of the partial amino acid sequences of several fragments of RC7-I, determined by protein chemical analyses, were found to be in excellent accordance with those deduced from the cDNA sequence. Computer analysis showed that RC7-I belongs to the beta-type subgroup of proteasomes with similarity to the beta-subunit of the archaebacterial proteasome, differing clearing from alpha-type subunits of the proteasome gene family. The overall structure of RC7-I was found to be homologous to that of yeast PRE1, which is necessary for chymotryptic activity.
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PMID:cDNA cloning of rat proteasome subunit RC7-I, a homologue of yeast PRE1 essential for chymotrypsin-like activity. 840 48

A comparative study of the chymotrypsin-like activity of the purified recombinant ClpP protease and the multicatalytic proteinase from rat liver is presented. The peptidase activity of both enzymes has been analyzed with several synthetic fluorogenic peptides, containing either aromatic or nonpolar amino acids in their P1 position. The respective Vmax, Km, and Vmax/Km were calculated from kinetic experiments. The substrate specificity of the multicatalytic proteinase, as expressed by Vmax/Km values, indicate the following substrate preference: N-Suc-IIW-MCA > N-Suc-LY-MCA > N-Suc-LLVY-MCA > or = N-Suc-AAF-MCA > N-Cbz-GGL-beta-NA > Glut-GGF-beta-NA > FPAM-4-MNA. In the case of the ClpP the order of preference is: N-Suc-LY-MCA > N-Suc-IIW-MCA > N-Suc-LLVY-MCA > or = N-Suc-AAF-MCA > or = N-Cbz-GGL-beta-NA > FPAM-4-MNA (where: N-Suc, N-succinyl-; MCA, 7-amido-4-methyl coumarin; beta-NA, beta-naphthylamide; N-Cbz, N-benzyloxycarbonyl-; 4-MNA, 4-methoxy-beta-naphthylamide; Glut, glutaryl. This similar substrate specificity is further supported by the lack of activity of both enzymes against SY-MCA and N-Suc-AAPF-MCA (known substrates of chymotrypsin), by very reduced activity against N-Suc-AAA-MCA and by no significant activity against LG-beta-NA. The results of mixed substrate experiments have shown that all the peptides that are substrates seem to be hydrolyzed by a single class of chymotrypsin-like site in both enzymes. The substrate specificity studies suggest a possible evolutionary relationship between the catalytic component of the ClpP of Escherichia coli and the multicatalytic proteinase chymotrypsin-like catalytic component. This conclusion is further supported by other circumstantial evidence: the fact that affinity-purified anti-ClpP antibodies cross-react with two polypeptide components of the rat liver multicatalytic proteinase complex, presented here and also shown previously; the known resemblance of both structures at the electron microscope level; and their reported role in the degradation of NH2-end rule substrates.
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PMID:A comparative study of the chymotrypsin-like activity of the rat liver multicatalytic proteinase and the ClpP from Escherichia coli. 840 53

The multicatalytic proteinase (MCP) complex is a major nonlysosomal proteinase which plays an important role in non-lysosomal pathways of protein degradation and which has recently been implicated in antigen processing. The mammalian MCP complex is composed of more than 20 different types of polypeptide, but it is not yet clear which of these components are responsible for its proteolytic activities. The complex has at least three distinct types of proteolytic activity. One of these, the so-called 'trypsin-like' activity, which involves cleavage on the carboxy side of basic amino acid residues, can be selectively and completely inhibited by peptidyl arginine aldehydes (such as leupeptin and antipain), and is also the most sensitive to inhibition by thiol-reactive reagents. In the present study N-[ethyl-1-14C]ethylmaleimide has been used to specifically label thiol groups protected by leupeptin binding. The results suggest that one or two polypeptide components within the complex can be protected against modification by N-ethylmaleimide. These components may be responsible for the 'trypsin-like' activity of the complex or may be adjacent to the catalytic component(s) and play an important role in substrate binding.
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PMID:Leupeptin-binding site(s) in the mammalian multicatalytic proteinase complex. 842 70

Ubiquitin is a highly conserved polypeptide found in all eukaryotes. The major function of ubiquitin is to target proteins for complete or partial degradation by a multisubunit protein complex called the proteasome. Here, the Drosophila fat facets gene, which is required for the appropriate determination of particular cells in the fly eye, was shown to encode a ubiquitin-specific protease (Ubp), an enzyme that cleaves ubiquitin from ubiquitin-protein conjugates. The Fat facets protein (FAF) acts as a regulatory Ubp that prevents degradation of its substrate by the proteasome. Flies bearing fat facets gene mutations were used to show that a Ubp is cell type--and substrate-specific and a regulator of cell fate decisions in a multicellular organism.
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PMID:Control of cell fate by a deubiquitinating enzyme encoded by the fat facets gene. 852 78

The biological effect of type 1 interferons is proposed to arise in part from the conjugation of ubiquitin cross-reactive protein (UCRP), the ISG15 gene product, to intracellular target proteins in a process analogous to that of its sequence homolog ubiquitin, a highly conserved 8.6-kDa polypeptide whose ligation marks proteins for degradation via the 26 S proteasome. Inclusion of CoCl2 during the purification of recombinant UCRP blocks the proteolytic inactivation of the polypeptide occurring by cleavage of the carboxyl-terminal glycine dipeptide required for activation and subsequent ligation. Intact UCRP supports a low rate of ubiquitin-activating enzyme (E1)-dependent ATP:PPi exchange but fails to form a stoichiometric E1-UCRP thiol ester or undergo transfer to ubiquitin carrier protein (E2). The binding affinity of E1 for UCRP is significantly diminished relative to that of ubiquitin. These results suggest that UCRP conjugation proceeds through an enzyme pathway distinct from that of ubiquitin, at least with respect to the step of activation. This was confirmed for an in vitro conjugation assay in which 125I-UCRP could be ligated in an ATP-dependent reaction to proteins present within an A549 human lung carcinoma cell extract and could be competitively inhibited by excess unlabeled UCRP but not ubiquitin. Other results demonstrate that 125I-UCRP conjugation is significantly increased in cell extracts after 24 h of incubation in the presence of interferon-beta, consistent with the late induction of UCRP conjugating activity. Thus, interferon-responsive cells contain a pathway for UCRP ligation that is parallel but distinct from that of ubiquitin.
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PMID:Conjugation of the 15-kDa interferon-induced ubiquitin homolog is distinct from that of ubiquitin. 855 May 81

Nascent polypeptide chains are in a dangerous situation as soon as they leave their place of birth, the channel of the large ribosomal subunit: more than 20 different pathways for the degradation of proteins exist in cells. Chaperones protect and guide the young protein molecules and support their correct foldings. Targeting signals direct the proteins to the organelles of their destination. The lysosome is the site of random degradation, while the proteasome is highly selective. Although these two organelles provide the most important pathways for the degradation of long- and short-lived proteins, other pathways with roles in deciding the fate of cellular proteins must also be considered.
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PMID:The fates of proteins in cells. 856 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

A previously identified intracellular proteolytic activity in the hyperthermophilic archaeon Pyrococcus furiosus (I. I. Blumentals, A. S. Robinson, and R. M. Kelly, Appl. Environ. Microbiol. 56:1992-1998, 1990) was found to be a homomultimer consisting of 18.8-kDa subunits. Dissociation of this native P. furiosus protease I (PfpI) into a single subunit was seen by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) but only after trichloroacetic acid precipitation; heating to 95 degrees C in the presence of 2% SDS and 80 mM dithiothreitol did not dissociate the protein. The gene (pfpI) coding for this protease was located in genomic digests by Southern blotting with probes derived from the N-terminal amino acid sequence. pfpI was cloned, sequenced, and expressed in active form in Escherichia coli as a fusion protein with a histidine tag. The recombinant protease from E. coli showed maximum proteolytic activity at 95 degrees C, and its half-life was 19 min at this temperature. This level of stability was significantly below that previously reported for the enzyme purified by electroelution of a 66-kDa band from SDS-PAGE after extended incubation of cell extracts at 98 degrees C in 1% SDS (>30 h). The pfpI gene codes for a polypeptide of 166 amino acid residues lacking any conserved protease motifs; no protease activity was detected for the 18.8-kDa PfpI subunit (native or recombinant) by substrate gel assay. Although an immunological relationship of this protease to the eukaryotic proteasome has been seen previously, searches of the available databases identified only two similar amino acid sequences: an open reading frame of unknown function from Staphylococcus aureus NCTC 8325 (171 amino acid residues, 18.6 kDa, 41% identity) and an open reading frame also of unknown function in E. coli (172 amino acid residues, 18.8 kDa, 47% identity). Primer extension experiments with P. furiosus total RNA defined the 5' end of the transcript. There are only 10 nucleotides upstream of the start of translation; therefore, it is unlikely that there are any pre- or pro-regions associated with PfpI which could have been used for targeting or assembly of this protease. Although PfpI activity appears to be the dominant proteolytic activity in P. furiosus cell extracts, the physiological function of PfpI is unclear.
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PMID:Sequence, expression in Escherichia coli, and analysis of the gene encoding a novel intracellular protease (PfpI) from the hyperthermophilic archaeon Pyrococcus furiosus. 862 29

A thermophilic Thermoactinomyces sp. E79 producing a highly thermostable alkaline protease was isolated from soil. The protease, produced extracellularly by Thermoactinomyces sp. E79, was purified by DEAE-Sepharose CL-6B and Butyl-Toyopearl 650M column chromatography. The relative molecular mass was estimated to be 31,000 by SDS-polyacrylamide gel electrophoresis. Enzyme activity was inhibited by phenylmethylsulfonyl fluoride, suggesting the enzyme to be a serine protease. The optimum temperature for the enzyme activity was 85 degrees C, and about 50% of the original activity remained after incubation at 90 degrees C for 10 min in the presence of Ca2+. The optimum pH for the enzyme activity was 11.0 and the enzyme was fairly stable from pH 5.0 to 12.0. The gene for this thermostable alkaline protease was cloned in Escherichia coli and the expressed intracellular enzyme was activated by heat treatment. Sequence analysis showed an open reading frame of 1,152 base pairs, coding for a polypeptide- of 384 amino acids. The polypeptide was composed of a signal sequence (25 amino acids), a prosequence (81 amino acids), and a mature protein of 278 amino acids. The deduced amino acid sequence of the mature protease had high similarity with thermitase, a serine protease from Thermoactinomyces vulgaris, and the extent of sequence identity was 76%.
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PMID:Purification and characterization of a thermostable alkaline protease from Thermoactinomyces sp. E79 and the DNA sequence of the encoding gene. 870 14

Molecular cloning of cDNA for a new regulatory subunit, designated p97, of the human 26S proteasome showed that the polypeptide consists of 908 amino acid residues with a calculated molecular mass of 100184 Da and an isoelectric point of 4.94. Computer analysis showed that p97 is very similar to type-1 tumor-necrosis-factor (TNF)-receptor-associated protein (TRAP)-2 and 55.11, both of which were identified recently as binding proteins of the cytoplasmic domain of type-1 TNF receptor by yeast two-hybrid screening. This finding suggests that the 26S proteasome might serve as a mediator molecule in the TNF signaling pathway in cells. Computer-assisted similarity analysis also revealed the high sequence similarity of p97 with a yeast protein whose function is yet unknown, the gene for which is here termed NAS1 (non-ATPase subunit 1). Disruption of NAS1 resulted in several phenotypes, including lethality and temperature-sensitive growth, depending on the genetic background of the cells used. The human p97 cDNA suppressed the growth defect of nas1 disruptant cells, when expressed from single-copy or multi-copy vectors, indicating that p97 is functionally equivalent to yeast Nas1p. Culturing of the temperature-sensitive nas1 cells at the restrictive temperature promoted the accumulation polyubiquitinated cellular proteins, implying that the 26S proteasome requires a functional Nas1p subunit for ubiquitin-dependent proteolysis. These results indicate that p97/Nas1p plays an important regulatory role in the function of the 26S proteasome.
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PMID:cDNA cloning and functional analysis of the p97 subunit of the 26S proteasome, a polypeptide identical to the type-1 tumor-necrosis-factor-receptor-associated protein-2/55.11. 877 43


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