pepstatin and 3-4-dichloroisocoumarin

Aspartic proteinase (EC 3.4.23) activity plays a pivotal role in the degradation of Theobroma cacao L. seed proteins during the fermentation step of cacao bean processing. Therefore, this enzyme is believed to be critical for the formation of the peptide and amino acid cocoa flavor precursors that occurs during fermentation. Using cDNA cloning and northern blot analysis, we show here that there are at least two distinct aspartic proteinase genes ( TcAP1 and TcAP2) expressed during cacao seed development. Both genes are expressed early during seed development and their mRNA levels decrease towards the end of seed maturation. TcAP2 is expressed at a much higher level than TcAP1, although the expression of TcAP1 increases slightly during germination. The proteins encoded by TcAP1 and TcAP2 are relatively different from each other (73% identity). This, and the fact that the two corresponding genes have different expression patterns, suggests that the TcAP1 and TcAP2 proteins may have different functions in the maturing seeds and during germination. Because the TcAP2 gene is expressed at a much higher level during seed development than TcAP1, it is likely that the TcAP2 protein is primarily responsible for the majority of the industrially important protein hydrolysis that occurs during cacao bean fermentation. Finally, TcAP2 has been functionally expressed in the yeast Yarrowia lipolytica. The secreted recombinant protein is able to hydrolyse bovine haemoglobin at acidic pH and is sensitive to pepstatin A, confirming that TcAP2 encodes an aspartic proteinase, and strongly suggests that this gene encodes the well-characterized aspartic proteinase of mature cacao seeds.

Topics: Amino Acid Sequence; Aspartic Acid Endopeptidases; Cacao; Cloning, Molecular; Coumarins; DNA, Complementary; Gene Expression Regulation, Enzymologic; Gene Expression Regulation, Plant; Hydrogen-Ion Concentration; Isocoumarins; Isoenzymes; Leucine; Molecular Sequence Data; Pepstatins; Phylogeny; Plant Proteins; RNA, Messenger; Seeds; Sequence Analysis, DNA; Sequence Homology, Amino Acid; Yarrowia

Glutamate carboxypeptidase II may modulate excitatory neurotransmission through the catabolism of the neuropeptide N-acetylaspartylglutamate (NAAG) and possibly other endogenous peptide substrates. To investigate the molecular properties of cloned human GCP II (hGCP II), we analyzed the NAAG-hydrolytic activity conveyed by transfection of a full-length hGCP II cDNA into PC3 cells, which do not express GCP II endogenously. Membrane fractions from these cells demonstrated activity with an apparent Km of 73 nM and Vmax of 35 pmol/(mg protein*min). Activity was inhibited by EDTA and stimulated by the addition of CoCl2. Addition of GCP II inhibitors beta-NAAG, quisqualic acid and 2-(phosphonomethyl)pentanedioic acid (PMPA) inhibited hydrolysis of 2.5 nM NAAG with IC50s of 201 nM, 155 nM and 98 pM, respectively. In competition experiments designed to infer aspects of hGCP II substrate selectivity, NAAG was the most potent alpha peptide tested, with an IC50 of 26 nM. Folate derivatives and some other gamma-glutamyl peptides showed comparable affinity to that of NAAG, also displaying IC50s in the low nM range. Taken together with previous evidence demonstrating their presence in GCP II-expressing tissues, these data suggest that both NAAG and folates are good candidate substrates for GCP II in vivo.

Topics: Animals; Antigens, Surface; Binding, Competitive; Carboxypeptidases; Chelating Agents; Cloning, Molecular; Cobalt; Coumarins; Dipeptides; Edetic Acid; Folic Acid; Glutamate Carboxypeptidase II; Histamine H1 Antagonists; Humans; Hydrolysis; Isocoumarins; Kinetics; Leucine; Male; Neuropeptides; Pepstatins; Phenanthrolines; Prostatic Neoplasms; Protease Inhibitors; Rats; Serine Proteinase Inhibitors; Substrate Specificity; Synaptic Transmission; Tumor Cells, Cultured

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