calcimycin and benzyloxycarbonylleucyl-leucyl-leucine-aldehyde

Endoplasmic reticulum (ER) stress can result in the accumulation of unfolded/misfolded protein in the ER lumen, which can trigger the unfolded protein response (UPR) resulting in the activation of various genes including immunoglobulin-binding protein (BiP; also known as glucose-regulated protein 78 or HSPA5). BiP, an ER heat shock protein 70 (HSP70) family member, binds to unfolded protein, inhibits their aggregation and re-folds them in an ATP-dependent manner. While cadmium, an environmental contaminant, was shown to induce the accumulation of HSP70 in vertebrate cells, less information is available regarding the effect of this metal on BiP accumulation or function. In this study, cadmium chloride treatment of Xenopus laevis A6 kidney epithelial cells induced a dose- and time-dependent increase in BiP, HSP70 and heme oxygenase-1 (HO-1) accumulation. Exposure of cells to a relatively low cadmium concentration at a mild heat shock temperature of 30°C greatly enhanced BiP and HSP70 accumulation compared to cadmium at 22°C. Treatment of cells with the glutathione synthesis inhibitor, buthionine sulfoximine, enhanced cadmium-induced BiP and HSP70 accumulation. Immunocytochemistry revealed that cadmium-induced BiP accumulation occurred in a punctate pattern in the perinuclear region. In some cells treated with cadmium chloride or the proteasomal inhibitor, MG132, large BiP complexes were observed that co-localized with aggregated protein or aggresome-like structures. These BiP/aggresome-like structures were also observed in cells treated simultaneously with cadmium at 30°C or in the presence of buthionine sulfoximine. In amphibians, the association of BiP with unfolded protein and its possible role in aggresome function may be vital in the maintenance of cellular proteostasis.

Topics: Animals; Buthionine Sulfoximine; Cadmium Chloride; Calcimycin; Cell Line; Dose-Response Relationship, Drug; Endoplasmic Reticulum Chaperone BiP; Environmental Pollutants; Epithelial Cells; Glutamate-Cysteine Ligase; Heat-Shock Proteins; Heat-Shock Response; Heme Oxygenase-1; HSP70 Heat-Shock Proteins; Kidney; Leupeptins; Oxidative Stress; Proteasome Inhibitors; Time Factors; Tunicamycin; Unfolded Protein Response; Up-Regulation; Xenopus laevis; Xenopus Proteins

Cyclooxygenase (COX)-1 and hematopoietic prostaglandin (PG) D synthase (H-PGDS) proteins, which are both involved in the arachidonate cascade, were stable in human megakaryocytic MEG-01 cells. In contrast, once the intracellular calcium level was increased by treatment with a calcium ionophore, both protein levels rapidly decreased with a half-life of less than 30 and 120 min for COX-1 and H-PGDS, respectively. In the presence of a proteasome inhibitor, COX-1 and H-PGDS proteins accumulated within 10 and 30 min, respectively, and concurrently appeared as the high-molecular-mass ubiquitinated proteins within 30 and 60 min, respectively, after an increase in the intracellular calcium level. The ubiquitination of these proteins was also observed when ADP, instead of a calcium ionophore, was used as an inducer to elevate the intracellular calcium level. When the entry of calcium ion into the cells was inhibited by ethylene glycol tetraacetic acid (EGTA), the ubiquitination of COX-1 and H-PGDS was clearly suppressed; and the addition of CaCl(2) to the medium cleared the EGTA-mediated suppression of the ubiquitination. These results indicate that COX-1 and H-PGDS were rapidly ubiquitinated and degraded through the ubiquitin-proteasome system in response to the elevation of the intracellular calcium level.

Topics: Adenosine Diphosphate; Arachidonic Acid; Calcimycin; Calcium Ionophores; Calcium Signaling; Cell Line; Cyclooxygenase 1; Half-Life; Humans; Intramolecular Oxidoreductases; Leupeptins; Lipocalins; Proteasome Endopeptidase Complex; Proteasome Inhibitors; Protein Stability; Proteolysis; Ubiquitinated Proteins; Ubiquitination

Transglutaminase 2 (TG2) is a calcium-dependent enzyme that catalyzes the transamidation reaction. There is conflicting evidence on the role of TG2 in apoptosis. In this report, we show that TG2 increases in response to low level of oxidative stress, whereas TG2 diminishes under high stress conditions. Monitoring TG2 expression, activity and calcium concentration in cells treated with A23187 revealed that the initial rise of calcium activates TG2 but subsequent calcium-overload induces the degradation of TG2 via calcium-mediated polyubiquitination. These results indicate that the role of TG2 in apoptosis depends on the level of calcium influx triggered by oxidative stress.

Topics: Adenoviridae; Anti-Bacterial Agents; Apoptosis; Calcimycin; Calcium; Cell Death; Cell Line; Culture Media, Serum-Free; Dose-Response Relationship, Drug; Enzyme Inhibitors; GTP-Binding Proteins; HeLa Cells; Humans; Kidney; Leupeptins; Oxidative Stress; Protein Glutamine gamma Glutamyltransferase 2; Time Factors; Transglutaminases; Ubiquitination

The membrane mobility agent, 2-(methoxyethoxy)ethyl-cis-8-(2-octylcyclopropyl)octanoate (A2C) promotes the fusion of rat, rabbit, and human erythrocytes in the presence of exogenous Ca2+. Under these conditions, the high sensitivity form of calcium-activated neutral protease (mu-calpain) in erythrocytes is activated autolytically. mu-Calpain is activated in accordance with fusion; that is, both erythrocyte fusion and autolytic activation of mu-calpain are induced in rat erythrocytes at 30 min, in rabbit erythrocytes at 150 min, and in human erythrocytes at 240 min after the addition of A2C and Ca2+. When erythrocytes are preincubated with the Ca2+ ionophore A23187, both fusion and autolytic activation start earlier. A leupeptin analogue, Cbz-Leu-Leu-Leu-aldehyde (ZLLLal), inhibits both the autolytic activation of mu-calpain and fusion induced by A2C and Ca2+. These results indicate that treatment of erythrocytes with A2C and Ca2+, results in first an influx of Ca2+ into the cells, followed by autolytic activation of mu-calpain, proteolysis of membrane proteins, exposure of fusion-sites, and, finally, fusion of erythrocytes.

Topics: Animals; Calcimycin; Calcium; Calpain; Enzyme Activation; Erythrocyte Membrane; Erythrocytes; Humans; Kinetics; Leupeptins; Membrane Fusion; Protease Inhibitors; Rabbits; Rats; Stearates