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)

A large, intracellular proteinase accumulated by inhibitors (PABI) was found in cultured mammalian cells as a large, multicatalytic proteinase with a greatly elevated concentration in the presence of small peptide proteinase inhibitors (Tsuji and Kurachi (1989) J. Biol. Chem. 264, 16093). Electron microscopic analysis showed that the tertiary structure of PABI highly resembled that of alpha 2-macroglobulin complexed with a proteinase(s). Isolation of the anti-PABI cross-reacting material from calf serum added to the culture media of baby hamster kidney cells further supported that the primary component of PABI was alpha 2-macroglobulin. Immunoblot analyses and the substrate specificity of PABI indicated that the major proteinase component contained in PABI was thrombin. When alpha 2-macroglobulin was added to the PABI-depleted serum, a significant accumulation or a degradation of the intracellular alpha 2-macroglobulin was observed in the presence or absence of leupeptin, respectively. Similarly, when thrombin was added to the PABI-depleted fetal calf serum supplemented with fresh alpha 2-macroglobulin, a significant amount of intracellular thrombin was found only in the presence of leupeptin. These results indicate that the major component of the intracellular PABI molecules is a complex of alpha 2-macroglobulin with thrombin which is internalized from the culture media. Intracellular accumulation of PABI, therefore, is a phenomenon primarily relevant to the culture cells. Whether or not PABI is also generated in certain physiological or pathological conditions requires further study.
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PMID:The major component of a large, intracellular proteinase accumulated by inhibitors is a complex of alpha 2-macroglobulin and thrombin. 171 Sep 31

Muscle protein degradation and intracellular protease activities were investigated in disseminated intravascular coagulation (DIC), which is frequently associated with severe catabolic states such as sepsis and multiple organ failure. DIC was introduced in rats by repeated intravenous thrombin injections. Saline was injected in control rats. In the 28 rats (14 with DIC and 14 controls), the bilateral soleus (SOL) muscles were incubated in an oxygenated medium without cycloheximide (CH) to determine the release of tyrosine (Tyr) into the incubated medium. From 24 rats (12 with DIC and 12 controls), the SOL and extensor digitorum longus (EDL) muscles were harvested to measure the activities of proteasome and of cathepsins L and B. The contralateral muscles were incubated in a medium with 0.5 mM CH to determine the release of Tyr and 3-methylhistidine (3-MH). The release of Tyr without CH (net proteolysis) from SOL muscles with DIC was greater than in controls (218 +/- 83.3 vs. 145 +/- 47.7 pmol/mg/h. However, the release of Tyr and 3-MH with CH (total proteolysis) and the activities of proteasome and cathepsins in DIC were nearly the same as those in controls. In both DIC and control rats, the total release of Tyr and proteasome activity were greater in SOL than in EDL muscles. These results suggest that reutilization of Tyr, reflecting protein synthesis, is suppressed in DIC and that the red slow muscle is more active in nonfibrillar proteolysis than the white fast muscle.
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PMID:Modulation of muscle protein metabolism in disseminated intravascular coagulation. 764 9

The isopenicillin-N-synthetase-encoding pcbC gene from the filamentous fungus Acremonium chrysogenum is differentially expressed in strains showing either a high or low cephalosporin C production. For a case study to demonstrate heterologous protein synthesis in A. chrysogenum, we have chosen a synthetic 195-bp gene encoding the thrombin inhibitor hirudin from the leech Hirudo medicinalis. The hirudin gene was fused with the 5' and 3' regions of the pcbC gene, resulting in four different expression vectors, which we named pHIR1 to pHIR4. In order to achieve secretion of the heterologous polypeptide, two out of four vectors carry, in addition, secretion signal sequences of an alkaline protease gene originating either from Fusarium sp. or from A. chrysogenum. After DNA-mediated transformation of the two A. chrysogenum strains, transformants were further analysed on the transcriptional and translational level. Irrespective of the vector used for transformation, all transformants show a hirudin-gene-specific transcript in Northern hybridizations. In further analysis, hirudin synthesis was determined with a thrombin-inhibition assay, but was detectable only in those strains carrying expression plasmids with the secretion signals. In this case, hirudin was secreted into the culture medium. Transformants from strains with a high cephalosporin C production showed a three- to eightfold higher expression of the hirudin gene compared to low cephalosporin-C-producing strains. The amount of recombinant hirudin was quantified further by ELISA and Western blotting, using a monoclonal antibody directed against recombinant hirudin. Finally, the time course of hirudin gene expression was investigated in a selected transformant that has hirudin activities of 8.0 ATU/ml culture medium. Northern hybridization experiments revealed the highest hirudin transcript level after 2-5 days of cultivation, showing the strongest signal after 3 days. After 4-5 days, we detected the highest hirudin activity, as was confirmed by Western blotting. The level of heterologous hirudin synthesis in A. chrysogenum is discussed in relation to other eukaryotic expression systems.
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PMID:Efficient synthesis of the blood-coagulation inhibitor hirudin in the filamentous fungus Acremonium chrysogenum. 927 48

Neurofilamentous conglomerates (NfCg), as axonal spheroids or conglomerates in motoneurons, are the histopathologic hallmarks for early stages of amyotrophic lateral sclerosis (ALS). We hypothesize that NfCg may be formed by post-translational modifications of altered Nf proteins that include: (1) hyperphosphorylation, (2) glycosylation (or glycoxidation), (3) nitration, (4) ubiquitination and/or (5) crosslinking by the Ca++-dependent transglutaminase (TGase). These, as well as other changes, are predicted to be initiated or accentuated by oxidative damage. The damaged Nf proteins then activate cascades of intracellular protein degradation which include ATP-dependent ubiquitin/proteasome proteolysis. Other proteolytic systems, either Ca++-dependent or independent, may also be activated, such as serine and cysteine protease systems. These enzymes, either lysosomal or non-lysosomal may also participate in the degradation of damaged Nf proteins being balanced by their cognate inhibitors. Protein complexes formed by these protease=inhibitor systems, along with damaged Nf proteins, may accumulate within the cell bodies as neuronal inclusions, since a number of intracellular inclusions are found in motor neurons in ALS. In the current study, we investigated the involvement of serine proteases and their serpins in NfCg formation. Pairs of three serine proteases (trypsin, chymotrypsin and thrombin) and their cognate serpins (alpha1-anti-trypsin, alpha1-anti-chymotrypsin, and protease nexin I) were probed in motoneurons with their antibodies for both NfCg and inclusions. Positive immunoreactivities for all serine proteases and their cognate serpins support the contention that the imbalance of serine proteases and internalized serpins may have a role in formation of NfCg and inclusions, and hence, the pathogenesis of ALS.
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PMID:Serpin=serine protease-like complexes within neurofilament conglomerates of motoneurons in amyotrophic lateral sclerosis. 985 54

Inherited antithrombin deficiency is associated with a predisposition for familial venous thromboembolic disease. Pleiotropic effect-type mutants of antithrombin that have an amino acid replacement in a distal hinge region including strands 1C, 4B, and 5B of the polypeptide chain are known to exhibit impaired interactions with both thrombin and heparin, coupled with a secretion defect. To examine the mechanism of pleiotropic effect-type antithrombin deficiency, we expressed three mutants, Oslo (Ala404-->Thr), Kyoto (Arg406-->Met), and Utah (Pro407-->Leu), in baby hamster kidney (BHK) cells, and compared their secretion rates, affinities for heparin and abilities to form thrombin-antithrombin (TAT) complexes with those of wild-type (Wt) antithrombin. Pulse-chase experiments showed that the Oslo- and Kyoto-mutants were secreted at rates similar to Wt antithrombin. In contrast, the Utah-mutant underwent partial intracellular degradation. The intracellular degradation of the Utah-mutant was not inhibited by lysosomotropic inhibitors, but by proteasome inhibitors such as carbobenzoxy-L-leucyl-L-leucyl-L-leucinal (LLL) and lactacystin, indicating that a part of the Utah-mutant was degraded by proteasome through quality control in the endoplasmic reticulum (ER). Crossed immunoelectrophoresis in the presence of heparin showed that only the Oslo-mutant lacks heparin-binding ability. Incubation with thrombin showed that the Kyoto- and Utah-mutants, but not the Oslo-mutant, formed a weak but detectable TAT complex. Furthermore, heparin enhanced the TAT complex formation by the Kyoto- and Utah-mutants, suggesting heparin cofactor activities of these mutants. These results show that each of the Oslo-, Kyoto-, and Utah-mutants exhibits different properties as to secretion, intracellular degradation and functional activity, although they are grouped as pleiotropic effect-type mutants.
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PMID:Cellular and functional characterization of three recombinant antithrombin mutants that caused pleiotropic effect-type deficiency. 999 Jan 21

Fertilization is a precisely controlled process involving many gamete molecules in sperm binding to and penetration through the extracellular matrix of the egg. After sperm bind to the extracellular matrix (vitelline coat), they undergo the acrosome reaction which exposes and partially releases a lytic agent called "lysin" to digest the vitelline coat for the sperm penetration. The vitelline coat sperm lysin is generally a protease in deuterostomes. The molecular mechanism of the actual degradation of the vitelline coat, however, remains poorly understood. In order to understand the lysin system, we have been studying the fertilization mechanism in ascidians (Urochordata) because we can obtain large quantities of gametes which are readily fertilized in the laboratory. Whereas ascidians are hermaphrodites, which release sperm and eggs simultaneously, many ascidians, including Halocynthia roretzi, are strictly self-sterile. Therefore, after sperm recognize the vitelline coat as nonself, the sperm lysin system is thought to be activated. We revealed that two sperm trypsin-like proteases, acrosin and spermosin, the latter of which is a novel sperm protease with thrombin-like substrate specificity, are essential for fertilization in H. roretzi. These molecules contain motifs involved in binding to the vitelline coat. We found that the proteasome rather than trypsin-like proteases has a direct lytic activity toward the vitelline coat. The target for the ascidian lysin was found to be a 70-kDa vitelline coat component called HrVC70, which is made up of 12 EGF-like repeats. In addition to the proteasome system, the ubiquitination system toward the HrVC70 was found to be necessary for ascidian fertilization. In this review, I describe recent progress on the structures and roles in fertilization of the two trypsin-like proteases, acrosin and spermosin, and also on the novel extracellular ubiquitin-proteasome system, which plays an essential role in the degradation of the ascidian vitelline coat.
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PMID:Ascidian sperm lysin system. 1201 76

In a proteasome-lacking mutant of Streptomyces coelicolor A3(2), an intracellular enzyme with chymotrypsin-like activity, absent from the wild type, was detected. Complementation that restored proteasome function did not suppress expression of the endopeptidase. Since the enzyme was not found in two other S. coelicolor proteasome mutants, its expression probably resulted from a secondary mutation arisen in the proteasome mutant. Purification of the endopeptidase revealed its identity to SCO7095, a putative hydrolase encoded by the S. coelicolor A3(2) genome with no known homologue. Based on the prediction of a Ser-Asp-His catalytic triad and an alpha/beta hydrolase fold, SCO7095 was assigned to peptidase clan SC. N-terminally His-tagged SCO7095 was efficiently expressed in Escherichia coli cells and purified for further characterization. Although SCO7095 is distantly related to several proline iminopeptidases, including Thermoplasma acidophilum tricorn-interacting F1, no aminopeptidase activity was detected. On synthetic substrates, the monomeric enzyme exhibited not only chymotrypsin-like activity but also thrombin-like activity.
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PMID:Characterization of a novel intracellular endopeptidase of the alpha/beta hydrolase family from Streptomyces coelicolor A3(2). 1251 96

Novel N-arylsulfonyldipeptidyl aldehyde derivatives were prepared by DMSO oxidation from the corresponding dipeptide alcohol, and their potencies as calpain inhibitors were evaluated in vitro. Among them, N-(4-fluorophenylsulfonyl)-l-valyl-l-leucinal (8, SJA6017) potently inhibited calpains. 8 also inhibited cathepsin B and L but did not inhibit other cysteine proteases (interleukin 1beta-converting enzyme), serine proteases (trypsin, chymotrypsin, thrombin, factor VIIa, factor Xa), or proteasome. Preliminary cytotoxicity studies of 8 exhibited a relatively safe profile.
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PMID:Structure-activity relationship study and drug profile of N-(4-fluorophenylsulfonyl)-L-valyl-L-leucinal (SJA6017) as a potent calpain inhibitor. 1259 66

Glycoprotein (GP) V is noncovalently linked to GPIbalpha, GPIbbeta and GPIX within the platelet GPIb-V-IX complex, a receptor for von Willebrand factor and thrombin. Two functions have been ascribed to GPV, namely, the modulation of thrombin- and collagen-dependent platelet responses. The biosynthesis of this molecule was investigated in pulse-chase metabolic labelling experiments performed in CHO cell lines transfected with GPV, alone or in the presence of GPIb-IX. GPV could not be detected at the surface of cells expressing the single subunit but was found instead as a soluble form in the culture medium. In pulse-chase studies, an immature 70 kDa protein was detected in cell lysates, whereas a fully processed 80-82 kDa form was only observed in the culture supernatants at later chase times. Immature GPV was N-glycosylated and retained before the medial Golgi while the secreted molecule contained complex sialylated sugars. The mature soluble form of GPV was produced by an enzymatic cleavage which was not affected by inhibitors of proteasome, calpain or metalloproteinases. When GPV was cotransfected with GPIb-IX, the former was no longer found in the culture supernatant but was retained in the cell membrane as shown by fluorescence-activated cell sorting and confocal microscopy analyses. Surface expressed GPV was processed from an immature 70 kDa form to produce a mature 80 kDa protein, processing similar to the intracellular trafficking of GPIbalpha. These results indicate that correct biosynthesis and surface expression of GPV in platelets requires the presence of the other subunits of the GPIb-V-IX complex.
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PMID:Biosynthesis of platelet glycoprotein V expressed as a single subunit or in association with GPIb-IX. 1535 44

The functional significance of protease-activated receptors (PARs) in endothelial cells is largely undefined, and the intracellular consequences of their activation are poorly understood. Here, we show that the serine protease thrombin, a PAR-1-selective peptide (TFLLRN), and SLIGKV (PAR-2-selective peptide) induce cyclooxygenase-2 (COX-2) protein and mRNA expression in human endothelial cells without modifying COX-1 expression. COX-2 induction was accompanied by sustained production of 6-keto-PGF1alpha, the stable hydrolysis product of prostacyclin, and this was inhibited by indomethacin and the COX-2-selective inhibitor NS398. PAR-1 and PAR-2 stimulation rapidly activated both ERK1/2 and p38MAPK, and pharmacological blockade of MEK with either PD98059 or U0126 or of p38MAPK by SB203580 or SB202190 strongly inhibited thrombin- and SLIGKV-induced COX-2 expression and 6-keto-PGF1alpha formation. Thrombin and peptide agonists of PAR-1 and PAR-2 increased luciferase activity in human umbilical vein endothelial cells infected with an NF-kappaB-dependent luciferase reporter adenovirus, and this, as well as PAR-induced 6-keto-PGF1alpha synthesis, was inhibited by co-infection with adenovirus encoding wild-type or mutated (Y42F) IkappaBalpha. Thrombin- and SLIGKV-induced COX-2 expression and 6-keto-PGF1alpha generation were markedly attenuated by the NF-kappaB inhibitor PG490 and partially inhibited by the proteasome pathway inhibitor MG-132. Activation of PAR-1 or PAR-2 promoted nuclear translocation and phosphorylation of p65-NF-kappaB, and thrombin-induced but not PAR-2-induced p65-NF-kappaB phosphorylation was reduced by inhibition of MEK or p38MAPK. Activation of PAR-4 by AYPGKF increased phosphorylation of ERK1/2 and p38MAPK without modifying NF-kappaB activation or COX-2 induction. Our data show that PAR-1 and PAR-2, but not PAR-4, are coupled with COX-2 expression and sustained endothelial production of vasculoprotective prostacyclin by mechanisms that depend on ERK1/2, p38MAPK, and IkappaBalpha-dependent NF-kappaB activation.
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PMID:Cyclooxygenase-2 induction and prostacyclin release by protease-activated receptors in endothelial cells require cooperation between mitogen-activated protein kinase and NF-kappaB pathways. 1646 9


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