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)

Madin Darby bovine kidney (MDBK) cells were used as a source to identify novel bovine chemotactic factors for granulocytes and monocytes. A major bovine granulocyte chemotactic protein (GCP-2) has previously been isolated. A novel bovine monocyte chemotactic protein (bo MCP) was produced on MDBK cells stimulated with phorbol ester. The 14-kDa protein was purified to homogeneity by adsorption to controlled pore glass, heparin affinity chromatography, cation-exchange FPLC, and RP-HPLC. The amino acid sequence of the NH2-terminally blocked protein was determined by Edman degradation using proteolytic fragments. The primary structure of the bo MCP, characterized by four conserved cysteines, allowed classification of the protein within the C-C chemokine family. Bo MCP-1B was most related to known human and bovine MCPs. Compared to bovine MCP-1 and MCP-2, the protein consists of 84% and 53% identical amino acids, respectively. Since this bo MCP was also most homologous to human and animal MCP-1, it was designated bo MCP-1B. The minimal effective dose of bo MCP-1B for monocyte chemotactic activity was 0.2 mM. The maximal migration index, reached at 2 nM, was comparable to that of natural human MCP-1. Furthermore, bo MCP-1B was found to be capable of stimulating beta-glucuronidase release from monocytes. In contrast, bo MCP-1B was not chemotactic for neutrophilic and eosinophilic granulocytes. By its biological and biochemical characteristics, bo MCP-1B has to be considered as an authentic additional MCP-1 chemokine. The existence of a possible human counterpart for this novel MCP-1B still needs to be elucidated.
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PMID:Purification, sequence analysis, and biological characterization of a second bovine monocyte chemotactic protein-1 (Bo MCP-1B). 794 49

The gene of subtilisin S41, an alkaline protease secreted by the psychrophile Bacillus TA41, encodes for a preproenzyme of 419 amino acids residues. The nucleotide sequence and NH2- and COOH-terminal amino acid sequencing of the purified enzyme indicate that the mature subtilisin S41 is composed of 309 residues with a predicted M(r) = 31,224. Subtilisin S41 shares most of its properties with mesophilic subtilisins (structure of the precursor, 52% amino acid sequence identity, alkaline pH optimum, broad specificity, Ca2+ binding) but is characterized by a higher specific activity on macromolecular substrate, by a shift of the optimum of activity toward low temperatures, and by a low thermal stability. The enzyme also differs by an acidic pI (5.3) and the presence of one disulfide bond. It is proposed that the psychrophilic enzyme possesses a more flexible molecular structure when compared to mesophilic and thermophilic subtilases in order to compensate for the reduction of reaction rates at low temperatures. The model of subtilisin S41 indeed reveals several features able to induce a more flexible, heat-labile conformation: the occurrence of four extended surface loops, a very hydrophilic surface through 11 extra Asp residues, and the lack of several salt bridges and aromatic-aromatic interactions. The low affinity of the Ca1 calcium binding site (Kd(app) = 10(-6) M), resulting possibly from one chelating side chain substitution and the stacking of Gly residues, also reflect a less compact conformation. The difference of free energy of stabilization between subtilisin S41 and a mesophilic subtilisin suggests that the balance of exo- and endothermically formed weak bonds is critical for the enzyme flexibility.
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PMID:Cold adaptation of proteins. Purification, characterization, and sequence of the heat-labile subtilisin from the antarctic psychrophile Bacillus TA41. 802 Dec 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

A novel human CC chemokine complementary DNA was identified in a library constructed from human fetal RNA, cloned into a baculovirus vector, and expressed in Sf9 insect cells. The mature recombinant protein that was released had the NH2-terminal sequence pyro-QPDALNVPSTC...and consisted of 75 amino acids. Minor amounts of two variants of 77 and 82 residues (NH2 termini: LAQPDA...and FNPQGLAQPDA...) were released as well. The novel chemokine was designated monocyte chemotactic protein 4 (MCP-4) and the variants were designated (LA)MCP-4 and (FNPQGLA)MCP-4. MCP-4 shares the pyroglutamic acidproline NH2-terminal motif and 56-61% sequence identity with the three known monocyte chemotactic proteins and is 60% identical to eotaxin. It has marked functional similarities to MCP-3 and eotaxin. Like MCP-3, MCP-4 is a chemoattractant of high efficacy for monocytes and T lymphocytes. On these cells, it binds to receptors that recognize MCP-1, MCP-3, and RANTES. On eosinophils, MCP-4 has similar efficacy and potency as MCP-3, RANTES, and cotaxin. It shares receptors with eotaxin and shows full cross-desensitization with this cosinophil-selective chemokine. Of the two variants, only (LA)MCP-4 could be purified in sufficient quantities for testing and was found to be at least 30-fold less potent than MCP-4 itself. This suggests that the 75-residue form with the characteristic NH2 terminus of an MCP is the biologically relevant species.
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PMID:Monocyte chemotactic protein 4 (MCP-4), a novel structural and functional analogue of MCP-3 and eotaxin. 864 49

Pseudomonas aeruginosa is a zinc metalloprotease which may be involved in many infection processes, especially in the lung. In order to evaluate the production of the enzyme in culture supernatants, we developed an assay using peptide derivatives; the conductimetric method was used for monitoring the enzymatic activities. Tetrapeptide derivatives were enzymatically synthesized by coupling Z-Ala2 and X-AlaR using either thermolysin or P. aeruginosa elastase itself. In these substrates, X could be phenylalanine, tyrosine, or leucine and C-protection was performed by either an amide (NH2) or a methyl (OMe) group. Z-Ala2-Phe-AlaNH2 was found to be the best substrate, giving a catalytic ratio kcat/KM of 8600 mM-1.s-1. The evaluation of the alkaline protease activity with this substrate showed that the catalytic ratio is 1000-fold lower. The sensitivity of the conductimetric method was also demonstrated with as little as 1 nM elastase (0.13 microgram), being easily and accurately detected (SD, 3.8% for 10 measurements). Furthermore, the enzymatic activity was measured in a culture supernatant from a clinical strain.
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PMID:Synthetic peptide substrates for a conductimetric assay of Pseudomonas aeruginosa elastase. 866 May 69

Interferon (IFN) gamma induces replacements of the proteasomal subunits X and Y by LMP7 and LMP2, respectively, resulting in an alteration of the proteolytic specificity. We found a third pair of proteasome subunits expressed reciprocally in response to IFN-gamma. Molecular cloning of a cDNA encoding one subunit designated as Z, downregulated by IFN-gamma, showed that it is a novel proteasomal subunit with high homology to MECL1, which is markedly induced by IFN-gamma. Thus, IFN-gamma induces subunit replacements of not only X and Y by LMP7 and LMP2, respectively, but also of Z by MECL1, producing proteasomes responsible for immunological processing of endogenous antigens. When processed from their precursors, three pairs of the 10 homologous, but distinct, beta-type subunits of eukaryotic proteasomes, that is, X/LMP7, Y/LMP2, and Z/MECL1, have an NH2-terminal threonine residue, assumed to be part of a catalytic center. These findings suggest that the altered molecular organization of the proteasome induced by IFN-gamma may be responsible for acquisition of its functional change.
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PMID:Newly identified pair of proteasomal subunits regulated reciprocally by interferon gamma. 866 37

We have studied the degradation of a set of long peptides (9-30 amino acids) from the nucleoprotein of influenza A. In common for all these peptides is the core sequence NH2-Ser-Arg-Tyr-Trp-Ala-Ile-Arg-Thr-Arg-COOH, NP383-391, known as an antigenic peptide specific for the HLA-B27 class I antigen. We show that this peptide is generated by enriched cytosolic proteasomes of two sizes, 20S and 12S. The 12S proteasome is the precursor, the preproteasome, to the 20S mature proteasome as shown by pulse-chase experiment and is most likely responsible for the proteolytic activity in the 12S region. Cleavage at the N-terminus is distinct and restricted to residue 383, independent of the N-terminal extension of the peptide. The C-terminus is generated via cleavage at three sites. Intermediate and final peptide products were identified by mass spectrometry. Finally, we show that the NP383-391 peptide generated by proteasomes in vitro is functional inasmuch as it possesses the ability to stimulate assembly of in vitro translated HLA-B27 antigens.
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PMID:Proteasomes generate in vitro a natural peptide of influenza-A nucleoprotein functional in HLA-B27 antigen assembly. 867 33

c-myb activation by insertional mutagenesis in murine myeloid leukemias can lead to amino (NH2)-terminal or carboxyl (COOH)-terminal truncation of its protein product. We observed that in these leukemias, the steady state level of the protein truncated at the COOH terminus was remarkably higher than that of the protein truncated at the NH2-terminus or full length wild-type protein. To examine the rate of proteolysis of different forms of Myb in a uniform cellular background, the proteins were constitutively expressed in the myeloblast cell line M1, using the retrovirus vector LXSN. In pulse chase experiments, using metabolically 35S-labeled proteins, it was determined that COOH-terminal truncation of c-Myb by 248 aa (CT-c-Myb) substantially increases protein stability, resulting in a t1/2 of about 140 min, as compared to 50 min for full length c-Myb (FL-c-Myb). In an investigation of the mechanism involved in the in vivo degradation of this short lived transcription factor, inhibitors of the lysosomal (chloroquine), proteasomal (ALLM, ALLN, lactacystin) and calpains (EGTA, E-64d, BAPTA/AM) pathways were utilized. Results of this experiment identified the 26S proteasome as a major pathway responsible for rapid breakdown of the protein in hematopoietic cells. Further experiments carried out in vitro demonstrated that c-Myb can be ubiquitinated, suggesting that this process may be involved in the targeting of wild-type c-Myb to degradation by the 26S proteasome. In addition, it was demonstrated that CT-c-Myb was less efficiently ubiquitinated than wild-type protein indicating that defects in modification account for its escape from rapid turnover. We speculate that the increased half-life of c-Myb resulting from truncation could contribute to its transforming potential.
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PMID:Oncogenic activation of c-Myb by carboxyl-terminal truncation leads to decreased proteolysis by the ubiquitin-26S proteasome pathway. 901 Feb 22

The proteasome is a multicatalytic protease complex that plays a key role in diverse cellular functions. The peptide vinyl sulfone, carboxybenzyl-leucyl-leucyl-leucine vinyl sulfone (Z-L3VS) covalently inhibits the trypsin-like, chymotrypsin-like and, unlike lactacystin, also the peptidylglutamyl peptidase activity in isolated proteasomes, and blocks their function in living cells. Although described as a class of mechanism-based inhibitors for cysteine proteases, the peptide vinyl sulfone Z-L3VS and a 125I-labeled nitrophenol derivative (125I-NIP-L3VS) covalently modify the active site threonine of the catalytic beta subunits of the proteasome. Modification of Thermoplasma proteasomes demonstrates the requirement for a hydroxyl amino acid (threonine, serine) as nucleophile at the beta subunit's NH2 terminus. 125I-NIP-L3VS covalently modifies the HslV subunit of the Escherichia coli protease complex HslV/HslU, a reaction that requires ATP, and supports a catalytic mechanism shared with that of the eukaryotic proteasome.
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PMID:Covalent modification of the active site threonine of proteasomal beta subunits and the Escherichia coli homolog HslV by a new class of inhibitors. 919 16

We isolated and sequenced a cDNA encoding mouse proteasome subunit LMP3 from a macrophage cDNA library. The gene encodes a 264-amino-acid protein with a calculated molecular mass of 29.11 kDa and an isoelectric point (pI) of 5.44. Comparison of the predicted protein sequence with that of the human and rat homologues, N3, revealed 11 and eight changes, respectively, in the cleaved NH2-terminal presequence of the precursor protein (pre-LMP3), and six and 10 changes, respectively, in the processed product. To corroborate the predicted molecular mass and pI, we analyzed LMP3 by immunoprecipitation with a mAb to human N3 that crossreacts with mouse LMP3. Precursor and processed forms of LMP3 were identified by 2D NEPHGE-PAGE, and their mobilities suggest the Lmp3 clone encodes the entire protein sequence.
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PMID:Cloning and characterization of mouse Lmp3 cDNA, encoding a proteasome beta subunit. 919 41


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