leupeptins and 3-4-dichloroisocoumarin

Proteasomes generate peptides that can be presented by major histocompatibility complex (MHC) class I molecules in vertebrate cells. Using yeast 20 S proteasomes carrying different inactivated beta-subunits, we investigated the specificities and contributions of the different beta-subunits to the degradation of polypeptide substrates containing MHC class I ligands and addressed the question of additional proteolytically active sites apart from the active beta-subunits. We found a clear correlation between the contribution of the different subunits to the cleavage of fluorogenic and long peptide substrates, with beta5/Pre2 cleaving after hydrophobic, beta2/Pup1 after basic, and beta1/Pre3 after acidic residues, but with the exception that beta2/Pup1 and beta1/Pre3 can also cleave after some hydrophobic residues. All proteolytic activities including the "branched chain amino acid-preferring" component are associated with beta5/Pre2, beta1/Pre3, or beta2/Pup1, arguing against additional proteolytic sites. Because of the high homology between yeast and mammalian 20 S proteasomes in sequence and subunit topology and the conservation of cleavage specificity between mammalian and yeast proteasomes, our results can be expected to also describe most of the proteolytic activity of mammalian 20 S proteasomes leading to the generation of MHC class I ligands.

Topics: Acetylcysteine; Amino Acid Sequence; Animals; Coumarins; Cysteine Endopeptidases; Fluorescence; Fungal Proteins; Histocompatibility Antigens Class I; Isocoumarins; Leupeptins; Molecular Sequence Data; Multienzyme Complexes; Peptides; Proteasome Endopeptidase Complex; Saccharomyces cerevisiae; Substrate Specificity; Vertebrates

To address the role of a plausible protease cascade in daunorubicin-triggered apoptosis, we evaluated the effect of cell-permeant protease inhibitors on its signal transduction pathway. Treatment of U937 and HL-60 cells with 0.5-1 microM of the chemotherapeutic drug daunorubicin induced a greater than 30% activation of neutral sphingomyelinase activity within 4-10 min with concomitant sphingomyelin hydrolysis and ceramide generation. DNA fragmentation and the classical morphological features of apoptosis were observed within 4-6 h. Pretreatment of cells with the serine protease inhibitors N-tosyl-L-phenylalanyl chloromethyl ketone (20 microM) or dichloroisocoumarin (20 microM) for 30 min inhibited daunorubicin-induced neutral sphingomyelinase activation, sphingomyelin hydrolysis, ceramide generation, and apoptosis. Other cell-permeant protease inhibitors such as pepstatin, leupeptin, and antipain had no such effect. The apoptotic response could be restored by the addition of 25 microM cell-permeant C6-ceramide. Daunorubicin-induced NF-kappaB activation was inhibited by dichloroisocoumarin but not by N-tosyl-L-phenylalanyl chloromethyl ketone, suggesting that this transcription factor can be activated independently of ceramide and is not directly implicated in the apoptotic pathway. These results suggest that inhibitors of serine proteases can act upstream of ceramide in drug-triggered apoptosis and that neutral sphingomyelinase activation is either directly or indirectly serine protease dependent.

Topics: Antibiotics, Antineoplastic; Antipain; Apoptosis; Ceramides; Coumarins; Daunorubicin; DNA Fragmentation; Dose-Response Relationship, Drug; Enzyme Activation; HL-60 Cells; Humans; Hydrolysis; Isocoumarins; Leupeptins; NF-kappa B; Pepstatins; Protease Inhibitors; Serine Proteinase Inhibitors; Signal Transduction; Sphingomyelin Phosphodiesterase; Sphingomyelins; Tosylphenylalanyl Chloromethyl Ketone; Tumor Cells, Cultured

The role of proteinases in renal proximal tubule (RPT) cellular death was examined using specific inhibitors of proteinases. Rabbit RPT suspensions were incubated with antimycin A for 1 h or tetrafluoroethyl-L-cysteine (TFEC) for 4 h in the absence or presence of the specific cysteine proteinase inhibitor L-trans-epoxysuccinyl-leucylamido (4-guanidino)butane (E-64), the serine proteinase inhibitors N-p-tosyl-L-lysine chloromethyl ketone (TLCK) or 3,4-dichloroisocoumarin (DCS), the serine and cysteine proteinase inhibitors leupeptin or antipain, or the aspartic proteinase inhibitor pepstatin. E-64 and pepstatin decreased lactate dehydrogenase (LDH) release, a marker of cell death, from RPT exposed either to antimycin A or TFEC. TLCK, DCS, leupeptin, or antipain did not decrease antimycin A- or TFEC-induced cell death. Bromohydroquinone- or t-butylhydroperoxide-induced cell death was not decreased by any of the proteinase inhibitors. Loss of lysosomal membrane potential, indicated by neutral red release, occurred prior to the onset of antimycin A-induced cell death. Extensive inhibition of lysosomal cathepsins B and L by E-64 was correlated with cytoprotection. However, E-64 was only protective after some cell death had occurred. These results suggest that lysosomal cysteine and aspartic proteinases, but not serine proteinases, play a role in RPT cell death induced by antimycin A or TFEC. The observation that E-64 was only protective after some cell death had occurred suggests that lysosomal cathepsins are released from dying cells and subsequently attack the remaining viable cells.

Topics: Animals; Anti-Bacterial Agents; Antimycin A; Antipain; Carboxypeptidases; Cathepsin A; Cathepsin B; Cell Death; Coumarins; Cysteine; Cysteine Proteinase Inhibitors; Dose-Response Relationship, Drug; Endopeptidases; Hydrocarbons, Fluorinated; Hydroquinones; Isocoumarins; Kidney Tubules, Proximal; L-Lactate Dehydrogenase; Leucine; Leupeptins; Lysosomes; Membrane Potentials; Pepstatins; Peroxides; Rabbits; Reactive Oxygen Species; Serine Proteinase Inhibitors; tert-Butylhydroperoxide; Tosyllysine Chloromethyl Ketone

Recent investigations indicate that proteolysis is an important event in generation of the apoptosis phenotype. Although various proteases have been suggested to be candidates for this proteolysis, the results from different laboratories are inconsistent. In the present studies, HL-60 cells were treated with cycloheximide to investigate proteases involved in apoptosis. The calpain inhibitors benzyloxycarbonyl-Leu-Leu-Tyr diazomethylketone and acetyl-Leu-Leu-Nle aldehyde were not capable of preventing apoptosis induced by cycloheximide. In the absence of cycloheximide, these two inhibitors could initiate apoptosis in HL-60 cells. The thiol protease inhibitor benzyloxycarbonyl-Leu-Val-Gly diazomethylketone neither prevented nor produced apoptosis. The serine protease inhibitors 3,4-dichloroisocoumarin (DCI) and tosyl-Phe chloromethylketone (TPCK) also induced apoptosis in the absence of cycloheximide. On the other hand, the latter two inhibitors decreased cycloheximide-induced apoptosis, assessed either by cell morphologic changes or DNA ladder generation. Benzyloxycarbonyl-Val-Ala-Asp fluoromethyl ketone and iodoacetamide, inactivators of interleukin 1beta-converting enzyme (ICE)-like proteases, did not produce apoptosis and inhibited the induction of apoptosis by cycloheximide, calpain inhibitors, or serine protease inhibitors. These results are consistent with the ICE-like proteases having a central role in proteolysis during apoptosis, while calpain-like proteases and the serine proteases sensitive to DCI or TPCK are not required for generation of the apoptosis phenotype in HL-60 cells.

Topics: Apoptosis; Calpain; Coumarins; Cycloheximide; Cysteine Proteinase Inhibitors; Diazomethane; HL-60 Cells; Humans; Isocoumarins; Leupeptins; Oligopeptides; Protein Synthesis Inhibitors; Serine Proteinase Inhibitors