cytochrome-c-t and 4-(2-aminoethyl)benzenesulfonylfluoride

The chaperone glucose-regulated protein, 78/immunoglobulin binding protein (GRP78/Bip), protects cells from cytotoxicity induced by DNA damage or endoplasmic reticulum (ER) stress. In this study, we showed that GRP78 is a major inducible protein in human non-small cell lung cancer H460 cells treated with ER stress inducers, including A23187 and thapsigargin. AEBSF, an inhibitor of serine protease, diminished GRP78 induction, enhanced mitochondrial permeability, and augmented apoptosis in H460 cells during ER stress. Simultaneously, AEBSF promoted Raf-1 degradation and suppressed phosphorylation of Raf-1 at Ser338 and/or Tyr340 during ER stress. Coimmunoprecipitation assays and subcellular fractionations showed that GRP78 associated and colocalized with Raf-1 on the outer membrane of mitochondria, respectively. While treatment of cells with ER stress inducers inactivated BAD by phosphorylation at Ser75, a Raf-1 phosphorylation site; AEBSF attenuated phosphorylation of BAD, leading to cytochrome c release from mitochondria. Additionally, overexpression of GRP78 and/or Raf-1 protected cells from ER stress-induced apoptosis. Taken together, our results indicate that GRP78 may stabilize Raf-1 to maintain mitochondrial permeability and thus protect cells from ER stress-induced apoptosis.

Topics: Apoptosis; bcl-Associated Death Protein; Cell Line; Cytochromes c; Endoplasmic Reticulum; Endoplasmic Reticulum Chaperone BiP; Heat-Shock Proteins; Humans; Membrane Potential, Mitochondrial; Mitochondria; Mitochondrial Membranes; Molecular Chaperones; Phosphorylation; Phosphoserine; Protein Binding; Protein Transport; Proto-Oncogene Proteins c-raf; Sulfones; Thapsigargin

Cells exposed to sustained endoplasmic reticulum (ER) stress undergo programmed cell death and display features typical of apoptosis, such as cysteine aspartyl protease (caspase) activation, cytochrome c release, and DNA fragmentation. Here, we show that the execution of cell death induced by ER stress is mediated via the proteasome. Inhibition of the proteasome by lactacystin prevented ER stress-induced degradation of Bcl-2, release of cytochrome c, processing of effector caspase-3, and exposure of phosphatidylserine. Owing to the ability of lactacystin to inhibit cytochrome c release, we propose that the pro-apoptotic activity of the proteasome lies upstream of mitochondrial activation. Thus, the proteasome serves as a principal mediator of ER stress-induced cell death in this system.

Topics: Acetylcysteine; Animals; Apoptosis; Blotting, Western; Brefeldin A; Caspase 3; Cells, Cultured; Chlorhexidine; Cytochromes c; DNA Fragmentation; Electrophoresis, Polyacrylamide Gel; Endoplasmic Reticulum; Fibroblasts; Mitochondrial Membranes; Models, Biological; Phosphatidylserines; Proteasome Endopeptidase Complex; Proteasome Inhibitors; Proto-Oncogene Proteins c-bcl-2; Rats; Sulfones; Temperature; Ubiquitin-Activating Enzymes

Bax and Bak are multidomain pro-apoptotic members of the Bcl-2 family of proteins that regulate mitochondria-mediated apoptosis by direct modulation of mitochondrial membrane permeability. Since double-knock-out mouse embryonic fibroblasts with deficiency of Bax and Bak are resistant to multiple apoptotic stimuli, Bax and Bak are considered to be an essential gateway for various apoptotic signals. Here we showed that the combination of calcium ionophore A23187 and arachidonic acid induced cytochrome c release and caspase-dependent death of double-knock-out mouse embryonic fibroblasts, indicating that other mechanisms of cytochrome c release exist. Furthermore, A23187/arachidonic acid (ArA)-induced caspase-dependent death was significantly suppressed by the treatment of several serine protease inhibitors including 4-(2-aminoethyl)benzenesulfonylfluoride and l-1-chloro-3-(4-tosylamido)-4-phenyl-2-butanone but not the overexpression of anti-apoptotic Bcl-2 family of proteins or the inhibition of mitochondrial membrane permeability transition. These results indicate that there are at least two mechanisms of cytochrome c release leading to caspase activation, a Bax/Bak-dependent mechanism and a Bax/Bak-independent, but serine protease(s)-dependent, mechanism.

Topics: Animals; Apoptosis; Arachidonic Acid; bcl-2 Homologous Antagonist-Killer Protein; bcl-2-Associated X Protein; Calcimycin; Caspases; Cell Membrane Permeability; Cells, Cultured; Cytochromes c; Ionophores; Mice; Mice, Knockout; Mitochondria; Serine Proteinase Inhibitors; Signal Transduction; Sulfones

The irreversible inhibitor of chymotrypsin-like serine proteases, N-tosyl-L-phenylalanine chloromethylketone (TPCK), was shown to prevent internucleosomal DNA cleavage caused by inducers of apoptosis. The pro-apoptotic properties of TPCK have been studied less thoroughly. The aim of the present study was to investigate the pro- and anti-apoptotic activities of TPCK on HL-60 cells and compare them with the actions of the mitochondrial electron transport inhibitor antimycin A (AMA). The results showed that TPCK alone caused activation of cell cycle checkpoints, mitochondrial cytochrome c release, caspase-3 activation, and chromatin condensation. Caspase-8 was not required for cytochrome c release but was crucial to caspase-3 activation. TPCK synergistically enhanced AMA-induced cytochrome c release and caspase-3 activation while completely blocking AMA-induced internucleosomal DNA fragmentation for at least 8 hours. Rather than blocking AMA-induced DNA fragmentation, the general serine protease inhibitor 4-(2-aminoethyl)-benzenesulphonyl fluoride (AEBSF) actually enhanced it. The pro-apoptotic effect of TPCK may be due to activation of cell cycle checkpoints via inhibition of the proteasome. The apoptotic pathways activated by TPCK and AMA probably converge at the level of the mitochondria. The mode by which TPCK prevents internucleosomal DNA fragmentation is probably not through serine protease inhibition.

Topics: Apoptosis; Caspase 3; Caspase 8; Caspase Inhibitors; Caspases; Cell Cycle; Cell Nucleus; Chymotrypsin; Cytochromes c; Enzyme Activation; Flow Cytometry; HL-60 Cells; Humans; Mitochondria; Serine Endopeptidases; Serine Proteinase Inhibitors; Sulfones; Tosylphenylalanyl Chloromethyl Ketone