carbobenzoxy-leucyl-leucyl-norvalinal and acetylleucyl-leucyl-norleucinal

ATF6, a 670 amino acid endoplasmic reticulum (ER) transmembrane glycoprotein with the electrophoretic mobility of a 90 kDa protein, is a key transcriptional activator of the unfolded protein response (UPR) that allows mammalian cells to maintain cellular homeostasis when the cells are subjected to a variety of environmental and physiological stress. Previous studies have established that ATF6 is a short-lived protein, the activation of which involves relocation from the ER to the Golgi where it is cleaved by the S1P/S2P protease system to generate a nuclear form that acts as a transcriptional activator for ER-stress inducible target genes such as Grp78/BiP. We report here that in addition to this process, ER-stress mediated by thapsigargin triggers an acute proteasomal degradation of the pre-existing pool of p90ATF6 independent of S1P/S2P cleavage. We showed that ATF6 is a direct target of proteasome-ubiquitin pathway, and this process can be suppressed by proteasome inhibitors, ALLN and MG115. We further observed that in non-stressed cells, p90ATF6 can be stabilized by MG115 but not ALLN and that treatment of cells with MG115 results in Grp78 induction in the absence of ER stress. These studies suggest that ER-stress induced acute, transit degradation of p90ATF6 could represent a novel cellular defense mechanism to prevent premature cell death resulting from p90ATF6 activation. Further, inhibition of proteasome activity can result in chaperone protein gene induction through stabilization of p90ATF6 as well as accumulation of malfolded proteins.

Topics: Activating Transcription Factor 6; Animals; CHO Cells; Cricetinae; Cysteine Proteinase Inhibitors; DNA-Binding Proteins; Endoplasmic Reticulum; Endoplasmic Reticulum Chaperone BiP; Enzyme Inhibitors; Heat-Shock Proteins; Humans; Kidney; Leupeptins; Mice; Molecular Chaperones; NF-kappa B; NIH 3T3 Cells; Protease Inhibitors; Proteasome Endopeptidase Complex; Thapsigargin; Transcription Factors

The cytokine IL-1beta suppresses rodent islets of Langerhans in vitro. Presently we used inhibitors of the proteasome to investigate if these compounds could counteract the suppressive effects of the cytokine. Thus, isolated rat islets were cultured and pre-treated with proteasome inhibitors and subsequently exposed for 48 h to 25 U/ml human IL-1beta. After this period functional tests were carried out. The rate of glucose oxidation (pmol/10 islets x 90 min) was suppressed by IL-1beta (115 +/- 17 vs. control 380 +/- 57). Pre-treatment with 10 microM of the proteasome inhibitor MG115 (N-carbobenzoxyl-leu-leu-norvalinal) and 100 microM of the calpain inhibitor norLEU (N-acetyl-leu-leu-norleucinal; known to affect proteasome activity) counteracted the suppressive effects (253 +/- 17 and 262 +/- 10 respectively). The calpain inhibitor alIMET (N-acetyl-leu-leu-methional) had no effect. MG115 (10 microM) and norLEU (100 microM) blocked nitric oxide formation induced by IL-1beta, while alIMET was without effect. We also investigated if IL-1beta could influence the expression of two inducible proteasome subunits, namely LMP2 and LMP7, and found that the cytokine increased the mRNA expression of the proteasome subunit LMP2 in islets, and that the proteasome inhibitor MG115 prevented this increase. In conclusion our study shows that IL-1beta increases the transcription of the proteasome subunit LMP2, and that the proteasome is involved in IL-1beta induced suppression of islet function. Moreover, the observation that inhibitors of the proteasome protect islets against IL-1beta induced inhibition of glucose metabolism, suggests that these compounds might be worthwile to explore in future therapies against the development of type 1 diabetes.

Topics: Animals; Base Sequence; Cysteine Endopeptidases; Cysteine Proteinase Inhibitors; DNA; DNA Primers; Electrophoretic Mobility Shift Assay; Glucose; Insulin; Insulin Secretion; Interleukin-1; Islets of Langerhans; Leupeptins; Male; Multienzyme Complexes; Nitrites; Proteasome Endopeptidase Complex; Rats; Rats, Sprague-Dawley; Reverse Transcriptase Polymerase Chain Reaction

1DNA topoisomerase II (topo II) is a nuclear enzyme that modifies DNA topology and also serves as a target to mediate the cytotoxicity of several antineoplastic agents. Several reports have demonstrated that a reduction of topo II is associated with reduced sensitivity to these agents. Topo II exists as two isoforms in mammalian cells: topo IIalpha and topo IIbeta. In MCF-7 cells, the half-life (mean +/- SEM) values of topo IIalpha and topo IIbeta in situ were 6.6 +/- 0.3 and 17.6 +/- 2.3 hr, respectively, as determined by [(35)S]methionine/cysteine pulse-chase analysis. Degradation of topo IIalpha in situ was abrogated by the presence of proteasome inhibitors, and the relative activities were carbobenzoxy-leucyl-leucyl-leucinal (MG132) > carbobenzoxy-leucyl-leucyl-norvalinal (MG115) > ALLN congruent with lactacystin. ATP-dependent degradation of topo IIalpha, but not topo IIbeta, was observed in extracts of asynchronously dividing HeLa and MCF-7 cells. Furthermore, degradation of topo IIalpha was abrogated by the proteasome inhibitors MG132 and MG115, but not by lactacystin, in extracts of asynchronously dividing MCF-7 cells. Finally, degradation of topo IIalpha, but not topo IIbeta, was observed to occur in a cell cycle-dependent fashion, in extracts of synchronized HeLa cells, with maximal loss of the alpha isoform occurring 2 hr after release from mitotic arrest. This degradation of topo IIalpha appeared to be facilitated by an ATP-dependent activity. Furthermore, high molecular weight bands (>200 kDa), which may represent polyubiquitinated-topo IIalpha conjugates, were also detected in extracts of synchronized HeLa cells. This study provides evidence for a role of the ubiquitin-proteasome pathway in the cell cycle-dependent regulation of topo IIalpha expression.

Topics: Acetylcysteine; Adenosine Triphosphate; Antigens, Neoplasm; Cell Cycle; Cell Extracts; Cysteine Endopeptidases; Cysteine Proteinase Inhibitors; DNA Topoisomerases, Type II; DNA-Binding Proteins; HeLa Cells; Humans; Isoenzymes; Leupeptins; Multienzyme Complexes; Proteasome Endopeptidase Complex; Tumor Cells, Cultured

Newly synthesized apolipoprotein B (apoB) is degraded by a proteolytic process in the pre-Golgi compartment that can be inhibited by N-acetyl-L-leucinyl-L-leucinyl-L-norleucinal (ALLN) but not by several other protease inhibitors. We have tested the hypothesis that the ubiquitin-proteasome pathway is involved in the intracellular degradation of apoB in liver cells. We found that inhibitors of proteasomes blocked the degradation of apoB in cultured human hepatoma (HepG2) cells. Protein degradation by proteasomes is ATP-dependent, and ATP depletion by dinitrophenol and 2-deoxyglucose also inhibited apoB degradation in these cells. Furthermore, the intracellular human apoB isolated by immunoprecipitation was shown to react specifically with anti-ubiquitin antibody by immunoblotting. This result was corroborated by sequential immunoprecipitation of [35S]methionine-labeled proteins by anti-human apoB and anti-ubiquitin antisera. In contrast, secreted apoB was not ubiquitinated. The amount of intracellular ubiquitinated apoB was increased by the proteasome inhibitors, ALLN and carbobenzoxyl-leucinyl-leucinyl-norvalinal-H (MG115). Our findings suggest that the ubiquitin-proteasome pathway is one mechanism for the intracellular degradation of apoB.

Topics: Adenosine Triphosphate; Antibodies; Apolipoproteins B; Blotting, Western; Cysteine Endopeptidases; Electrophoresis, Polyacrylamide Gel; Endoplasmic Reticulum; Leupeptins; Liver; Models, Biological; Multienzyme Complexes; Precipitin Tests; Protease Inhibitors; Proteasome Endopeptidase Complex; Tumor Cells, Cultured; Ubiquitins

Multiple cell adhesion proteins are up-regulated in vascular endothelial cells in response to TNF alpha and other inflammatory cytokines. This increase in cell adhesion gene expression is thought to require the transcription factor NF-kappa B. Here, we show that peptide aldehyde inhibitors of the proteasome, a multicatalytic protease recently shown to be required for the activation of NF-kappa B, block TNF alpha induction of the leukocyte adhesion molecules E-selectin, VCAM-1, and ICAM-1. Striking functional consequences of this inhibition were observed in analyses of leukocyte-endothelial interactions under defined flow conditions. Lymphocyte attachment to TNF alpha-treated endothelial monolayers was totally blocked, while neutrophil attachment was partially reduced but transmigration was essentially prevented.

Topics: Base Sequence; Calpain; Cell Adhesion; Cell Adhesion Molecules; Cells, Cultured; Cysteine Endopeptidases; E-Selectin; Endothelium, Vascular; Gene Expression; Humans; In Vitro Techniques; Intercellular Adhesion Molecule-1; Interleukin-8; Leukocytes; Leupeptins; Molecular Sequence Data; Multienzyme Complexes; NF-kappa B; Oligodeoxyribonucleotides; Proteasome Endopeptidase Complex; Proto-Oncogene Proteins; RNA, Messenger; Time Factors; Transcription Factor RelB; Transcription Factors; Tumor Necrosis Factor-alpha; Vascular Cell Adhesion Molecule-1

Reagents that inhibit the ubiquitin-proteasome proteolytic pathway in cells have not been available. Peptide aldehydes that inhibit major peptidase activities of the 20S and 26S proteasomes are shown to reduce the degradation of protein and ubiquitinated protein substrates by 26S particles. Unlike inhibitors of lysosomal proteolysis, these compounds inhibit the degradation of not only abnormal and short-lived polypeptides but also long-lived proteins in intact cells. We used these agents to test the importance of the proteasome in antigen presentation. When ovalbumin is introduced into the cytosol of lymphoblasts, these inhibitors block the presentation on MHC class I molecules of an ovalbumin-derived peptide by preventing its proteolytic generation. By preventing peptide production from cell proteins, these inhibitors block the assembly of class I molecules. Therefore, the proteasome catalyzes the degradation of the vast majority of cell proteins and generates most peptides presented on MHC class I molecules.

Topics: Amino Acid Sequence; Animals; Antigen Presentation; B-Lymphocytes; Cysteine Endopeptidases; Dose-Response Relationship, Drug; Hematopoietic Stem Cells; Histocompatibility Antigens Class I; Leupeptins; Lymphocytes; Mice; Molecular Sequence Data; Multienzyme Complexes; Ovalbumin; Protease Inhibitors; Proteasome Endopeptidase Complex; T-Lymphocytes; Ubiquitins

We demonstrate an essential role for the proteasome complex in two proteolytic processes required for activation of the transcription factor NF-kappa B. The p105 precursor of the p50 subunit of NF-kappa B is processed in vitro by an ATP-dependent process that requires proteasomes and ubiquitin conjugation. The C-terminal region of p105 is rapidly degraded, leaving the N-terminal p50 domain. p105 processing can be blocked in intact cells with inhibitors of the proteasome or in yeast with proteasome mutants. These inhibitors also block the activation of NF-kappa B and the rapid degradation of I kappa B alpha induced by tumor necrosis factor alpha. Thus, the ubiquitin-proteasome pathway functions not only in the complete degradation of polypeptides, but also in the regulated processing of precursors into active proteins.

Topics: Adenosine Triphosphate; Amino Acid Sequence; Animals; Cells, Cultured; Cysteine Endopeptidases; DNA-Binding Proteins; Humans; I-kappa B Proteins; Leupeptins; Models, Biological; Molecular Sequence Data; Multienzyme Complexes; NF-kappa B; NF-kappa B p50 Subunit; NF-KappaB Inhibitor alpha; Protease Inhibitors; Proteasome Endopeptidase Complex; Protein Precursors; Protein Processing, Post-Translational; Ubiquitins