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 mechanism of the activation of intracellular proteasomes at fertilization was measured in living sand dollar eggs using the membrane-impermeant fluorogenic substrate, succinyl-Phe-Leu-Arg-coumarylamido-4-methanesulfonic acid. When the substrate was microinjected into unfertilized eggs, the initial velocity of hydrolysis of the substrate (V0) was low. V0 measured 5 to 10 min after fertilization was five to nine times the prefertilization level and remained high throughout the first cell cycle. Hydrolysis of the substrate was inhibited by clasto-lactacystin beta-lactone, a specific inhibitor of the proteasome. There has been in vitro evidence that calcium may be involved in regulation of proteasome activity to either inhibit the increase in peptidase activity associated with PA 28 binding to the 20S proteasome or stimulate activity of the PA 700-proteasome complex. Since both intracellular free Ca2+ concentration ([Ca2+]i) and intracellular pH (pHi) increase after fertilization, hydrolysis of the proteasome substrate was measured under conditions in which [Ca2+]i and pHi were varied independently during activation. When the pHi of unfertilized eggs was elevated by exposure to 15 mM ammonium chloride in pH 9 seawater, V0 increased to a level comparable to that measured after fertilization. In contrast, [Ca2+]i elevation without pHi change, induced by calcium ionophore in sodium-free seawater, had no effect on V0 in the unfertilized egg. Moreover, when unfertilized eggs were microinjected with buffers modulating pHi, V0 increased in a pH-dependent manner. These results indicate that the pHi rise at fertilization is the necessary prerequisite for activation of the proteasome, an essential component in the regulation of the cell cycle.
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PMID:Activation of the proteasomes of sand dollar eggs at fertilization depends on the intracellular pH rise. 1020 42

A membrane proteinase from Pseudomonas aeruginosa, called insulin-cleaving membrane proteinase (ICMP), was located in the outer membrane leaflet of the cell envelope. The enzyme is expressed early in the logarithmic phase parallel to the bacterial growth during growth on peptide rich media. It is located with its active center facing to the outermost side of the cell, because its whole activity could be measured in intact cells. The very labile membrane proteinase was solubilized by non-ionic detergents (Nonidet P-40, Triton X-100) and purified in its amphiphilic form to apparent homogeneity in SDS-PAGE by copper chelate chromatography and two subsequent chromatographic steps on Red-Sepharose CL-4B (yield 58.3%, purification factor 776.3). It consisted of a single polypeptide chain with a molecular mass of 44.6 kDa, determined by mass spectrometry. ICMP was characterized to be a metalloprotease with pH-optimum in the neutral range. The ICMP readily hydrolyzed Glu(13)-Ala(14) and Tyr(16)-Leu(17) bonds in the insulin B-chain. Phe(25)-Tyr(26) and His(10)-Leu(11) were secondary cleavage sites suggesting a primary specificity of the enzyme for hydrophobic or aromatic residues at P'(1)-position. The ICMP differed from elastase, alkaline protease and LasA in its cleavage specificity, inhibition behavior and was immunologically diverse from elastase. The amino acid sequence of internal peptides showed no homologies with the known proteinases. This outer membrane proteinase was capable of specific cleavage of alpha and beta fibrinogen chains. Among the p-nitroanilide substrates tested, substrates of plasminogen activator, complement convertase and kallikrein with arginine residues in the P(1)-subsite were the substrates best accepted, but they were only cleaved at a very low rate.
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PMID:Characterization and purification of an outer membrane metalloproteinase from Pseudomonas aeruginosa with fibrinogenolytic activity. 1045 58

Because parenteral feeding is associated with negative N balance and reduced rates of protein synthesis in intestinal mucosa, we hypothesized that luminal exposure to specific amino acids or energy fuels would stimulate intestinal protein synthesis. We studied the acute effects of luminal nutrients on mucosal protein synthesis in the absence of systemic influences. Multiple jejunal segments constructed in piglets deprived of food overnight (n = 6) were randomly assigned to luminal perfusion with saline, 30 mmol/L amino acid mixture with or without 50 mmol/L glucose, or 30 mmol/L glutamine for 90 min. Protein synthesis was then measured by luminal perfusion with L-[2,6-(3)H]-phenylalanine. Energy substrates (glucose, short-chain fatty acids or beta-hydroxybutyrate) had no effect on mucosal protein synthesis. Relative to saline, a 30 mmol/L amino acid mixture or 30 mmol/L glutamine suppressed mucosal protein synthesis by 20-25% (P < 0.05). On the basis of these surprising results, we speculated that a coordinate reduction of proteolytic processes would be required to maintain positive intestinal N balance. Although intestinal protein catabolism cannot be assessed directly, the 30 mmol/L amino acid mixture acutely suppressed mucosal levels of mRNA encoding ubiquitin, 14-kDa ubiquitin conjugating enzyme and the C9 subunit of the proteasome by 20-30% (P < 0.05), demonstrating the sensitivity of components of the ATP-ubiquitin proteolytic pathway to acute regulation by nutrients. The suppression of protein synthesis by luminal amino acids in the absorptive state might lower intestinal utilization of amino acids to ensure efficient allocation of absorbed nutrients to nonintestinal tissues.
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PMID:Luminal amino acids acutely decrease intestinal mucosal protein synthesis and protease mRNA in piglets. 1049 61

Subtilisin Carlsberg, an alkaline protease from Bacillus licheniformis, was modified with polyoxyethylene (PEG) or aerosol-OT (AOT), and the solubility, conformation, and catalytic activity of the modified subtilisins in some organic media were compared under the same conditions. The solubility of modified subtilisins depended on the solubility of the modifier. On the other hand, the conformational changes depended on the solubility, rather than the property, of the modifier. When the modified subtilisin was dissolved in water-miscible polar solvents such as dimethylsulfoxide, acetonitrile, and tetrahydrofuran, significant conformational changes occurred. When modified subtilisin was dissolved in water-immiscible organic solvents, such as isooctane and benzene, the solvent did not induce significant conformational changes. The catalytic activity in the transesterification reaction of the N-acetyl-L-phenylalanine ethylester of the modified subtilisin in organic solvents was higher than that of native subtilisin. The high activity of modified subtilisin was thought to be due to a homogeneous reaction by the dissolved enzymes.
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PMID:Catalytic activity and conformation of chemically modified subtilisin Carlsberg in organic media. 1057 97

Micrococcus luteus isolated from human skin secretes an alkaline protease which degrades elastin. M. luteus protease (MLP) was produced in the late logarithmic and stationary phases of growth. MLP, purified to homogeneity by a three-step process, had a molecular mass of 32,812 Da and an isoelectric point of 9.3. MLP was active and highly stable in solution for 24 h from pH 6.0 to 10.5; it had maximal activity at temperatures between 57 and 59 degrees C. The presence of calcium in the solution was essential for enzyme activity and to prevent autolysis. Optimal activity occurred between pH 9.0 and 9.5, with 60% maximal activity from pH 6.5 to 11.0. The enzyme was inhibited by the serine enzyme inhibitors phenylmethylsulfonyl fluoride and chymostatin but not by the metalloenzyme inhibitor 1,10-phenanthroline or sulfhydryl enzyme inhibitors. Casein, bovine serum albumin, ovalbumin, beta-lactoglobulin, and elastin were digested by the protease while collagen and keratin were resistant to digestion. MLP demonstrated both esterase and amidase activity on synthetic peptide substrates. MLP preferentially cleaved the Leu(15)-Tyr(16) and Phe(24)-Phe(25) bonds of the oxidized beta-chain of insulin. Longer digests of insulin and the pattern of activity against synthetic substrates suggest that MLP has a cleavage specificity for bulky, hydrophobic, or aromatic amino acids in the P(1) or P(1)' positions. Amino acid sequences from the N-terminus and internal peptides of MLP were unique.
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PMID:Purification and characterization of a unique alkaline elastase from Micrococcus luteus. 1064 68

N(alpha)-acetylation, catalyzed co-translationally with N(alpha)-acetyltransferase (NAT), is the most common modifications of eukaryotic proteins. In yeast, there are at least three NATs: NAT1, MAK3, and NAT3. The 20 S proteasome subunits were purified from the normal strain and each of the deletion mutants, nat1, mak3, and nat3. The electrophoretic mobility of these subunits was compared by two-dimensional gel electrophoresis. Shifts toward the alkaline side of the gel and unblocking of the N terminus of certain of the subunits in one or another of the mutants indicated that the alpha1, alpha2, alpha3, alpha4, alpha7, and beta3 subunits were acetylated with NAT1, the alpha5 and alpha6 subunits were acetylated with MAK3, and the beta4 subunit was acetylated with NAT3. Furthermore, the Ac-Met-Phe-Leu and Ac-Met-Phe-Arg termini of the alpha5 and alpha6 subunits, respectively, extended the known types of MAK3 substrates. Thus, nine subunits were N (alpha)-acetylated, whereas the remaining five were processed, resulting in the loss of the N-terminal region. The 20 S proteasomes derived from either the nat1 mutant or the normal strain were similar in respect to chymotrypsin-like, trypsin-like, and peptidylglutamyl peptide hydrolyzing activities in vitro, suggesting that N(alpha)-acetylation does not play a major functional role in these activities. However, the chymotrypsin-like activity in the absence of sodium dodecyl sulfate was slightly higher in the nat1 mutant than in the normal strain.
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PMID:N(alpha)-acetylation and proteolytic activity of the yeast 20 S proteasome. 1067 91

Proteasomes are large multisubunit proteinases which have several distinct catalytic sites. In this study a series of di- and tri-peptidyl boronic acids have been tested on the chymotrypsin-like activity of purified mammalian 20 S and 26 S proteasomes assayed with succinyl-Leu-Leu-Val-Tyr-amidomethylcoumarin (suc-Leu-Leu-Val-Tyr-AMC) as substrate. The inhibition of 20 S proteasomes is competitive but only slowly reversible. The K(i) values for the best inhibitors were in the range 10-100 nM with suc-Leu-Leu-Val-Tyr-AMC as substrate, but the compounds tested were much less effective on other proteasome activities measured with other substrates. Free boronic acid inhibitors exhibited equivalent potency to their pinacol esters. Both benzoyl (Bz)-Phe-boroLeu and benzyloxycarbonyl (Cbz)-Leu-Leu-boroLeu pinacol ester inhibited 20 S and 26 S proteasomes with non-ideal behaviour, differences in inhibition of the two forms of proteasomes becoming apparent at high inhibitor concentrations (above 3xK(i)). Both of these compounds were also potent inhibitors of 20 S and 26 S proteasomes in cultured cells. However, gel filtration of cell extracts prepared from cells treated with radiolabelled phenacetyl-Leu-Leu-boroLeu showed that only 20 S proteasomes were strongly labelled, demonstrating differences in the characteristics of inhibition of 20 S and 26 S proteasomes. The usefulness of peptidyl boronic acid inhibitors for investigations of proteasome-mediated protein degradation was confirmed by the observation that Bz-Phe-boroLeu and Cbz-Leu-Leu-boroLeu pinacol ester inhibited NFkappaB activation with IC(50) values comparable to their K(i) values for purified proteasomes. The latter result supports the view that the chymotrypsin-like activity of proteasomes assayed with suc-Leu-Leu-Val-Tyr-AMC is a critical one for protein degradation in cells.
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PMID:Characterization of peptidyl boronic acid inhibitors of mammalian 20 S and 26 S proteasomes and their inhibition of proteasomes in cultured cells. 1067 65

The Saccharomyces cerevisiae nuclear gene RPM2 encodes a component of the mitochondrial tRNA-processing enzyme RNase P. Cells grown on fermentable carbon sources do not require mitochondrial tRNA processing activity, but still require RPM2, indicating an additional function for the Rpm2 protein. RPM2-null cells arrest after 25 generations on fermentable media. Spontaneous mutations that suppress arrest occur with a frequency of approximately 9 x 10(-6). The resultant mutants do not grow on nonfermentable carbon sources. We identified two loci responsible for this suppression, which encode proteins that influence proteasome function or assembly. PRE4 is an essential gene encoding the beta-7 subunit of the 20S proteasome core. A Val-to-Phe substitution within a highly conserved region of Pre4p that disrupts proteasome function suppresses the growth arrest of RPM2-null cells on fermentable media. The other locus, UMP1, encodes a chaperone involved in 20S proteasome assembly. A nonsense mutation in UMP1 also disrupts proteasome function and suppresses Deltarpm2 growth arrest. In an RPM2 wild-type background, pre4-2 and ump1-2 strains fail to grow at restrictive temperatures on nonfermentable carbon sources. These data link proteasome activity with Rpm2p and mitochondrial function.
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PMID:Proteasome mutants, pre4-2 and ump1-2, suppress the essential function but not the mitochondrial RNase P function of the Saccharomyces cerevisiae gene RPM2. 1075 50

During recent years, it has become increasingly clear that the ubiquitin-proteasome proteolytic pathway regulates intracellular protein degradation in various physiological and pathophysiological conditions. Substrates specifically degraded by the proteasome are important tools to assess the involvement of the proteasome in cellular proteolysis. It was recently proposed that the membrane permeable substrate methoxysuccinyl-phenylalanine-leucine-phenylalanine-7-amido-4- trifluoromethyl coumarin (FLF) is degraded specifically by the proteasome. The role of other proteolytic pathways in the degradation of FLF, however, is not fully understood. In the present study, we tested the role of different proteolytic pathways in the degradation of FLF in cultured myotubes and HepG2 cells by treating the cells with inhibitors of lysosomal, calpain and proteasome activity. In addition, we tested the hypothesis that insulin blocks proteasome-dependent degradation of FLF in myotubes and HepG2 cells. Results suggest that degradation of FLF in both myotubes and HepG2 cells is regulated by proteasome and calpain activity but not by lysosomal activity. Insulin inhibited proteasome-dependent but not calpain-dependent degradation of FLF in both myotubes and HepG2 cells. The results are important because they suggest that FLF degradation does not specifically reflect proteasome activity.
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PMID:Degradation of methoxysuccinyl-phe-leu-phe-7-amido-4-trifluoromethyl coumarin (FLF) in cultured myotubes and HepG2 cells is proteasome- and calpain/calcium-dependent. 1078 64

The peptide boronic acid derivative 2-Pyz-(CO)-Phe-Leu-B(OH)(2) is a potent inhibitor of 20S proteasome and a proposed anticancer agent. During preformulation studies, the compound presented erratic stability behavior. Efforts were made to isolate and identify the degradation products, thereby helping to identify possible mechanisms for the degradation. The reaction of 2-Pyz-(CO)-Phe-Leu-B(OH)(2) with hydrogen peroxide not only provided a convenient way to isolate the initial degradation products seen from hydrolysis in aqueous buffers but also showed that the major, initial degradation pathway was probably oxidative in nature. The isolated degradation products were characterized by nuclear magnetic resonance spectroscopy, mass spectrometry, and optical rotation dispersion. In the presence of hydrogen peroxide, the boronic acid group was cleaved from 2-Pyz-(CO)-Phe-Leu-B(OH)(2) to give an alcohol with an apparent retention of the original stereochemistry. Subsequent isomerization and further hydrolysis were then seen. Surprisingly, added ascorbate and EDTA accelerated rather than inhibited degradation. Degradation of 2-Pyz-(CO)-Phe-Leu-B(OH)(2) under acidic and basic conditions seemed to be mediated by an initial oxidative degradation pathway similar to that seen with the peroxide.
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PMID:Degradation pathways of a peptide boronic acid derivative, 2-Pyz-(CO)-Phe-Leu-B(OH)(2). 1082 34


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