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

---

Query: EC:3.4.25.1 (proteasome)
28,817 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

The aim of the present study was to characterize human CYP2E1 turnover and examine the possible proteolytic pathways responsible for the rapid degradation of CYP2E1 in a transfected HepG2 cell line expressing human CYP2E1. Two methods were used to study the CYP2E1 turnover; after addition of cycloheximide, the half-life of the CYP2E1 in the intact cells was about 6 h as detected by PNP catalytic activity assay and immunoblot analysis of apoprotein content. CYP2E1 substrates or ligands such as 4-methylpyrazole, ethanol, glycerol, and dimethyl sulfoxide protected CYP2E1 against this rapid degradation, whereas CCl4 accelerated this process. The second procedure involved pulse-chase experiments after labeling CYP2E1 with [35S]methionine and immunoprecipitation with anti-human CYP2E1 IgG. The half-life of CYP2E1 was about 2.5 h, and the various substrates or ligands modified the turnover process within intact cells as described for the cycloheximide experiments. More than 20 different reagents including antioxidants, physiological metabolites, lysosomal inhibitors, and protease inhibitors were screened for possible effects on CYP2E1 proteolytic degradation. Dibutyryl cAMP had no effect on CYP2E1 activity or turnover. Among those reagents tested so far, the serine protease inhibitor 1-chloro-3-tosylamido-7-amino-2-heptanone hydrochloride exhibited some protection against CYP2E1 degradation. To demonstrate whether the proteasome complex is involved in this process, Czb-Ile-Glu(OtBu)-Ala-leucinal (PSI) as a cell penetrating aldehydic proteasome inhibitor and Czb-Leu-norleucinal (calpeptin inhibitor) as an aldehydic nonproteosomal protease inhibitor were used to examine their effect on both the normal and the CCl4-stimulated CYP2E1 proteolytic degradation pathways. Treatment with PSI at concentrations ranging from 5 to 80 microM resulted in a dose-dependent protection against the loss of both the normal CYP2E1 and the CCl4-modified CYP2E1. The maximum protection by PSI at a concentration of 80 microM after a 12-h chase period was about 60% in cells treated with 2 mM CCl4 or 75% in cells without CCl4 treatment. Calpeptin inhibitor afforded little or no protection against CYP2E1 degradation in the absence or presence of CCl4. PSI did not inhibit CYP2E1 catalytic activity, suggesting that it was not a ligand for CYP2E1. These results indicate that human CYP2E1 has a short half-life span and that substrates can significantly modify its turnover rate in intact HepG2 cells. The proteasome proteolytic pathway may be involved in the degradation process of both the normal and the CCl4-modified human CYP2E1 in this model.
...
PMID:Characterization of cytochrome P4502E1 turnover in transfected HepG2 cells expressing human CYP2E1. 914 49

We studied the effect of inhibition of microsomal triglyceride transfer protein (MTP) on apolipoprotein (apo) B100 translation and secretion using HepG2 cells. The MTP-mediated lipid transfer activity was reduced using a specific MTP inhibitor. ApoB100 translation was synchronized by treatment with puromycin prior to L-[35S]methionine pulse-chase labeling. During the first 4 min of chase, synthesis of apoB polypeptides the size of 100-200 kDa was insensitive to the inhibitor, suggesting that inhibition of MTP did not affect the initiation of apoB100 translation. After 15 min of chase, the 100-200-kDa species were chased into polypeptides larger than 320 kDa (i.e. apoB65 or 65% of full-length apoB100) in both control and inhibitor-treated cells. However, the amount of these polypeptides decreased (by 36% for apoB65-75, by 64% for apoB75-85, by 76% for apoB85-95, and by 77% for apoB100) upon MTP inhibition. No accumulation of smaller polypeptides was observed, but total immunoprecipitable apoB radioactivity was decreased suggesting that apoB could undergo co-translational degradation when MTP activity was reduced. Inhibitors of the multicatalytic proteinase complex (proteasome) such as lactacystin or MG-115 could prevent apoB co-translational degradation. Nevertheless, MG-115 could not avoid the MTP inhibitor decreasing apoB100 secretion but rather induced the accumulation of secretion-incompetent apoB100 in the cell. These results indicate that MTP activity is required during the elongation of apoB100 polypeptides, particularly at the sequences downstream of carboxyl terminus of apoB65. Co-translational degradation might constitute a more general mechanism of early quality control for large or complex proteins.
...
PMID:Co-translational degradation of apolipoprotein B100 by the proteasome is prevented by microsomal triglyceride transfer protein. Synchronized translation studies on HepG2 cells treated with an inhibitor of microsomal triglyceride transfer protein. 925 52

The proteasome inhibitors, lactacystin and N-acetyl-leucyl-leucyl-norlucinal, caused a rapid and near-complete loss of approximately 22-23-kDa ubiquitinated nucleoproteins, which we have identified as monoubiquitinated nucleosomal histones H2A and H2B by immunological and two-dimensional electrophoretic techniques. In human SKBr3 breast tumor cells, depletion of monoubiquitinated histones by the proteasome inhibitors coincided with the accumulation of high molecular weight ubiquitinated proteins in both nucleoprotein and cytosolic fractions and decreased unconjugated ubiquitin in the cytosol, without changes in the nonubiquitinated core histones. Unconjugated ubiquitin was not detected in isolated tumor cell nuclei. A similar loss in monoubiquitinated histones occurred in cells harboring a defective, temperature-sensitive mutation of the ubiquitin-activating E1 enzyme, after these cells were elevated from 33 degrees C to the non-permissive temperature of 39 degrees C. DNA replication and RNA transcription were decreased by the proteasome inhibitors most strongly after 90% of the ubiquitin had been removed from ubiquitinated histones H2A and H2B, suggesting a relationship between the nucleosomal histone ubiquitin status and the processing of genetic information. Interestingly, although both proteasome inhibitors caused a generalized decrease in methionine incorporation into proteins, they strongly induced the synthesis of the hsp72 and hsp90 stress proteins. Finally, treating cells with heat-shock at 43 degrees C, with stress response-provoking chemicals or with several other proteasome inhibitors caused ubiquitinated proteins to accumulate, depleted free ubiquitin, and concomitantly decreased nucleosomal monoubiquitinated histones. These results suggest that deubiquitination of nucleosomal histones H2A and H2B may play a previously unrecognized role in the cellular stress response, as well as in the processing of chromatin, and emphasize the important role of the proteasome in cellular homeostasis.
...
PMID:Rapid deubiquitination of nucleosomal histones in human tumor cells caused by proteasome inhibitors and stress response inducers: effects on replication, transcription, translation, and the cellular stress response. 939 60

An immunological hierarchy among three H-2Db-restricted cytotoxic T lymphocyte (CTL) determinants in simian virus 40 (SV40) large T antigen (Tag) was described previously: determinants I and II/III are immunodominant, whereas determinant V is immunorecessive. To assess the immunogenicity of each determinant individually and define mechanisms that contribute to the immunorecessive nature of determinant V, we constructed a panel of recombinant vaccinia viruses (rVVs) expressing minigenes encoding these determinants in various polypeptide contexts. We found the following. (i) Immunization of mice with an rVV encoding full-length SV40 Tag resulted in priming for CTL responses to determinants I and II/III but not determinant V. (ii) rVVs encoding peptide I or II/III in the cytosol or targeted to the endoplasmic reticulum (ER) were highly antigenic and immunogenic. (iii) rVVs encoding peptide V minigenes were antigenic and immunogenic if the peptide was targeted to the ER, expressed in the cytosol with short flanking sequences, or expressed from within a self-protein, murine dihydrofolate reductase. (iv) Presentation of the nonflanked peptide V (preceded by a Met codon only) could be enhanced by using a potent inhibitor of the proteasome. (v) H-2Db-epitope V peptide complexes decayed more rapidly than complexes containing epitope I or II/III peptides. In brefeldin A blocking experiments, functional epitope V complexes were detected longer on targets expressing ER-targeted epitope V than on targets expressing forms of epitope V dependent on the transporter associated with antigen processing. Therefore, limited formation of relatively unstable cell surface H-2Db complexes most likely contributes to the immunorecessive nature of epitope V within SV40 Tag. Increasing the delivery of epitope V peptide to the major histocompatibility complex class I presentation pathway by ER targeting dramatically enhanced the immunogenicity of epitope V.
...
PMID:An endoplasmic reticulum-targeting signal sequence enhances the immunogenicity of an immunorecessive simian virus 40 large T antigen cytotoxic T-lymphocyte epitope. 944 50

Fluorescence emission properties of the alkaline protease Esperase have been investigated using steady-state and time-resolved fluorescence spectroscopy. The local polarity and solvent accessibility of the tryptophyl chromophores is characterized. Quenching studies demonstrated that Trp 6 and Trp 113 are 'buried' to acrylamide, iodide ions and caesium ions. An abnormally low tryptophan quantum yield was calculated showing that the emission of the two indole rings is significantly quenched by nearby side chains or peptide bonds. The fluorescence decay of PMS-Esperase was well fitted by two exponentials with lifetimes of 2.7 and 0.35 ns. X-ray data for Esperase (S. Klupsch, Ph.D. Thesis, University of Hamburg, Hamburg, Germany) in the region of the two tryptophans were used to explain the observed emission properties. Gln 182 and Asn 204 as well as Asn 117 and Met 119 are the most likely quenchers, respectively, of the Trp 6 and Trp 113 fluorescence. The two tryptophans in Esperase are 'buried' in hydrophobic regions and are excellent intrinsic probes to study folding-unfolding reactions. Experiments in the presence and absence of added calcium ions demonstrated the stabilizing role of the Ca(2+)-binding sites.
...
PMID:Steady-state and time-resolved fluorescence of Esperase: comparison with the X-ray structure in the region of the two tryptophans. 969 45

A major portion of newly synthesized apolipoprotein B (apoB) is degraded intracellularly. This degradation has been demonstrated to be mediated largely by the ubiquitin-proteasome pathway. We examined whether nascent apoB polypeptides or full-length apoB is selectively retrotranslocated from the endoplasmic reticulum into the cytosol for degradation. Herein, we found that full-length apoB as well as partial-length apoB peptides are ubiquitinated in HepG2 cells, and ubiquitination is an exclusively cytosolic process. Calnexin, which binds specifically to glycoproteins, has been postulated to promote apoB folding and complete translocation; we found that ubiquitinated apoB is bound to calnexin, suggesting that ubiquitinated apoB is glycosylated. In addition to calnexin binding, we have other pieces of evidence that the full-length intracellular ubiquitinated apoB is glycosylated, because (i) it binds to concanavalin A, and (ii) glycan can be demonstrated in the full-length ubiquitinated apoB by a chemical detection method involving oxidation of adjacent hydroxyl groups in the glycan moiety. Because glycosylation occurs inside the endoplasmic reticulum, the full-length glycosylated apoB must have been retrotranslocated into the cytosol for ubiquitination and proteasome-mediated degradation. Next we synchronized translation in HepG2 cells by puromycin treatment. A pulse-chase experiment using [35S]methionine labeling of intracellular apoB in these synchronized cells demonstrated that nascent partial-length apoB peptides are also ubiquitinated cotranslationally. We conclude that the ubiquitin proteasome-mediated degradation of apoB targets both nascent peptides cotranslationally before translocation as well as full-length apoB after its translocation into the endoplasmic reticulum.
...
PMID:Proteasome-mediated degradation of apolipoprotein B targets both nascent peptides cotranslationally before translocation and full-length apolipoprotein B after translocation into the endoplasmic reticulum. 976 44

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.
...
PMID:Cellular and functional characterization of three recombinant antithrombin mutants that caused pleiotropic effect-type deficiency. 999 Jan 21

MHC class I molecules display peptides selected from a poorly characterized pool of peptides available in the endoplasmic reticulum. We analyzed the diversity of peptides available to MHC class I molecules by monitoring the generation of an OVA-derived octapeptide, OVA257-264 (SL8), and its C-terminally extended analog, SL8-I. The poorly antigenic SL8-I could be detected in cell extracts only after its conversion to the readily detectable SL8 with carboxypeptidase Y. Analysis of extracts from cells expressing the minimal precursor Met-SL8-I by this method revealed the presence of SL8/Kb and the extended SL8-I/Kb complexes, indicating that the peptide pool contained both peptides. In contrast, cells expressing full length OVA generated only the SL8/Kb complex, demonstrating that the peptide pool generated from the full length precursor contained only a subset of potential MHC-binding peptides. Deletion analysis revealed that SL8-I was generated only from precursors lacking additional C-terminal flanking residues, suggesting that the generation of the C terminus of the SL8 peptide involves a specific endopeptidase cleavage. To investigate the protease responsible for this cleavage, we tested the effect of different protease inhibitors on the generation of the SL8 and SL8-I peptides. Only the proteasome inhibitors blocked generation of SL8, but not SL8-I. These findings demonstrate that the specificities of the proteases in the Ag-processing pathway, which include but are not limited to the proteasome, limit the diversity of peptides available for binding by MHC class I molecules in the endoplasmic reticulum.
...
PMID:Specific proteolytic cleavages limit the diversity of the pool of peptides available to MHC class I molecules in living cells. 1020 12

We sequenced five peptides from subunit 11 (S11), a 43 kDa protein of the human 26S proteasome, and used this information to clone its cDNA. The S11 cDNA encodes a 376 amino acid protein with a pI of 5.6 and a molecular mass of 42.9 kDa. Translation of S11 RNA in the presence of [35S]methionine produces a radiolabeled protein that co-migrates with S11 of the human 26S proteasome on SDS-PAGE. Polyclonal antiserum made against recombinant S11 recognizes a protein of the same size in extracts of bacteria expressing S11 and in purified 26S proteasomes from human red blood cells or rabbit reticulocytes. The S11 sequence does not contain motifs that suggest a biological function. S11 is, however, the human homolog of Rpn9, a recently identified subunit of the yeast 26S proteasome.
...
PMID:Identification, molecular cloning, and characterization of subunit 11 of the human 26S proteasome. 1022 35

Amino acid residues in the NH(2)-terminal region (Glu(2) - Ala(14)) of adult fast twitch skeletal muscle sarcoplasmic reticulum Ca(2+)-ATPase (SERCA1a) were deleted or substituted, and the mutants were expressed in COS-1 cells. Deletion of any single residue in the Ala(3)-Ser(6) region or deletion of two or more consecutive residues in the Ala(3)-Thr(9) region caused strongly reduced expression. Substitution mutants A4K, A4D, and H5K also showed very low expression levels. Deletion of any single residue in the Ala(3)-Ser(6) region caused only a small decrease in the specific Ca(2+) transport rate/mg of SERCA1a protein. In contrast, other mutants showing low expression levels had greatly reduced specific Ca(2+) transport rates. In vitro expression experiments indicated that translation, transcription, and integration into the microsomal membranes were not impaired in the mutants that showed very low expression levels in COS-1 cells. Pulse-chase experiments using [(35)S]methionine/cysteine labeling of transfected COS-1 cells demonstrated that degradation of the mutants showing low expression levels was substantially faster than that of the wild type. Lactacystin, a specific inhibitor of proteasome, inhibited the degradation accelerated by single-residue deletion of Ala(3). These results suggest that the NH(2)-terminal region (Ala(3) -Thr(9)) of SERCA1a is sensitive to the endoplasmic reticulum-mediated quality control and is thus critical for either correct folding of the SERCA1a protein or stabilization of the correctly folded SERCA1a protein or both.
...
PMID:Deletions or specific substitutions of a few residues in the NH(2)-terminal region (Ala(3) to Thr(9)) of sarcoplasmic reticulum Ca(2+)-ATPase cause inactivation and rapid degradation of the enzyme expressed in COS-1 cells. 1044 57


<< Previous 1 2 3 4 5 6 7 8 9 10 Next >>

---