alpha-chymotrypsin and succinyl-leucyl-leucyl-valyl-tyrosyl-methylcoumarinamide

ATP- and ubiquitin-independent proteolysis by the 20S proteasome is responsible for the selective degradation of oxidized proteins. In vitro, the 20S proteasome shows an increased proteolytic activity toward oxidized polypeptides and the suc-LLVY-MCA peptide specific for its chymotrypsin-like activity. We have analyzed the effect of the intracellular redox status on the chymotrypsin-like activity of the 20S proteasome in human T47D cells overexpressing the detoxifiant enzyme seleno-glutathione peroxidase-1 (GPx-1). We report a 30% decreased activity of the chymotrypsin-like activity in cells overexpressing GPx-1. This phenomenon correlated with a 2-fold increase in IkappaB alpha half-life, a protein whose basal turnover is 20S proteasome-dependent. Following exposure to H2O2, these cells showed a seleno-dependently decreased accumulation of intracellular reactive oxygen species and 20S proteasome chymotrypsin-like activity. Similar results were obtained in HeLa cells transiently overexpressing human GPx-1. Moreover, exposure of HeLa cells to antioxidant compounds reduced the proteasome 20S chymotrypsin-like activity. In contrast, no effects were observed when HeLa cell extracts used to determine proteasome activity were incubated with either reduced or oxidized glutathione. These results suggest that GPx-1 activity or pro-reducing conditions can downregulate basal 20S proteasome activity. Hence, the intracellular redox status, probably through the level of oxidized proteins, is an important element that can either activate or down-regulate the 20S proteasome chymotrypsin-like activity in living cells.

Topics: Antioxidants; Chymotrypsin; Coumarins; Cysteine Endopeptidases; Electrophoresis, Polyacrylamide Gel; Gene Expression Regulation, Enzymologic; Glutathione Peroxidase; Glutathione Peroxidase GPX1; HeLa Cells; Humans; Immunoblotting; Indicators and Reagents; Multienzyme Complexes; Oligopeptides; Oxidation-Reduction; Precipitin Tests; Proteasome Endopeptidase Complex; Reactive Oxygen Species

Earlier studies have demonstrated that human platelets contain the 20S proteasome, and its protein activator. However, understanding the potential role of the proteasome in human platelets requires a detailed knowledge about its chymotryptic-like activity, a crucial one for protein degradation in all eukaryotic cells. In this communication we have shown that human platelet 20S proteasome exhibited chymotryptic-like activity towards succinyl-Leu-Leu-Val-Tyr-7-amido-4-methylcoumarin as substrate at a broad pH range, with optimum between pH 7.5-8.0 and 5.0-5.5. These two activities were markedly inhibited by a 10 micromol/l concentration of two structurally unrelated proteasome inhibitors: lactacystin/beta-lactone or benzyloxycarbonyl-Ile-Glu(O-tert.-butyl)-Ala-leucinal, but not by ebelactone B, an inhibitor of lysosomal cathepsin A/deamidase. The chymotryptic-like activity of the 20S proteasome against succinyl-Leu-Leu-Val-Tyr-7-amido-4-methylcoumarin was also significantly inhibited in platelets, after exposure of platelet-rich plasma to 10 micromol/l lactacystin and benzyloxycarbonyl-Ile-Glu(O-tert.-butyl)-Ala-leucinal for up to 60 min. This indicates that these inhibitors can enter platelets and selectively inhibit 20S proteasome activity. We also demonstrated for the first time by Western blot analysis that human platelets contain a proteasome activator, PA28, which is known to play a key role in antigen processing by significant stimulation of the proteasomal chymotryptic-like activity. Since the platelet 20S proteasome was also present in a latent form, this suggests that its activity may be regulated in vivo in human platelets. All these results can therefore be beneficial in future studies on the role of the 20S proteasome in platelet biology.

Topics: Acetylcysteine; Blood Platelets; Chymotrypsin; Coumarins; Cysteine Endopeptidases; Enzyme Activation; Enzyme Inhibitors; Humans; Hydrogen-Ion Concentration; Multienzyme Complexes; Oligopeptides; Proteasome Endopeptidase Complex

When tested on Suc-Leu-Leu-Val-Tyr-MCA as substrate, purified full-length hsp90 displays a low "chymotrypsin-like" peptidase activity which is activated by Ca++ and Mg++ ions. On the other hand, using long-term in vitro experiments, we demonstrate the ability of hsp90 to convert into a 73 kDa truncated product. This autocatalytic degradation proceeds from the C-terminal end of the full-length hsp90 and shifts the oligomers toward monomeric truncated forms. This corresponds to an intermolecular process as addition of exogenous 73 kDa product speeds up the maturation kinetics. The peptidase activity is enhanced in the 73 kDa product and is sensitive to peptide aldehyde inhibitors but only partially to lactone compounds. The degradation process itself presents a great degree of similarity with the peptidase activity toward either the inhibitors or the tested ions. Neither 20S proteasome nor m-calpain are responsible for the observed activities. Indeed, the self-processing is a consequence of the peptidase activity which appears to be an intrinsic property of the chaperone. The functional importance of these findings is discussed.

Topics: Animals; Calcium; Calpain; Catalysis; Cations, Divalent; Cattle; Chymotrypsin; Copper; Coumarins; Cysteine Endopeptidases; Dimerization; Electrophoresis, Polyacrylamide Gel; Enzyme Stability; HSP90 Heat-Shock Proteins; Kinetics; Magnesium; Multienzyme Complexes; Muscle, Skeletal; Oligopeptides; Peptide Fragments; Peptide Hydrolases; Protease Inhibitors; Proteasome Endopeptidase Complex; Protein Processing, Post-Translational; Rabbits; Sodium Dodecyl Sulfate; Zinc

The fungal epipolythiodioxopiperazine metabolite gliotoxin has a variety of toxic effects such as suppression of antigen processing, induction of macrophagocytic apoptosis and inhibition of transcription factor NF-kappaB activation. How gliotoxin acts remains poorly understood except that the molecule's characteristic disulfide bridge is important for immunomodulation. As this fungal metabolite stabilizes the NF-kappaB inhibitor IkappaBalpha in the cytoplasm, we decided to investigate its molecular mechanism of action.. We show that gliotoxin is an efficient, noncompetitive inhibitor of the chymotrypsin-like activity of the 20S proteasome in vitro. Proteasome inhibition can be reversed by dithiothreitol, which reduces gliotoxin to the dithiol compound. In intact cells, gliotoxin inhibits NF-kappaB induction through inhibition of proteasome-mediated degradation of IkappaBalpha.. Gliotoxin targets catalytic activities of the proteasome efficiently. Inhibition by gliotoxin may be countered by reducing agents, which are able to inactivate the disulfide bridge responsible for the inhibitory capacity of gliotoxin.

Topics: Cells, Cultured; Chymotrypsin; Coumarins; Cysteine Endopeptidases; DNA-Binding Proteins; Gliotoxin; HeLa Cells; Humans; I-kappa B Proteins; Monocytes; Multienzyme Complexes; NF-kappa B; NF-KappaB Inhibitor alpha; Oligopeptides; Proteasome Endopeptidase Complex; Tumor Necrosis Factor-alpha

Synthetic inhibitors of the multicatalytic proteinase complex (proteasome) can provide the means to uncover the functional significance and catalytic mechanism of this macromolecule. Although inhibitor development is still in its early stages, some useful compounds have already been prepared. Of the various types of inhibitors thus far studied, peptidyl aldehydes have been the most effective. Since peptidyl aldehydes inhibit both serine and cysteine proteinases, lack of specificity is their major limitation. The properties of one such compound N-benzyloxycarbonyl-IE(Ot-Bu)A-Leucinal, a potent inhibitor of suc-LLVY-MCA hydrolysis, are described in detail.

Topics: Amino Acid Sequence; Animals; Cell Line; Chymotrypsin; Coumarins; Cysteine Endopeptidases; Cysteine Proteinase Inhibitors; Mice; Molecular Sequence Data; Multienzyme Complexes; Oligopeptides; Proteasome Endopeptidase Complex; Substrate Specificity; Ubiquitins

Our previous study suggested that a chymotrypsin-like protease was involved in the motility of chum salmon sperm (Inaba K, Morisawa M, Biomed Res (1991) 12, 435-437). In this study, we examined the peptidase activity of demembranated sperm of chum salmon using ten synthetic peptides. When spermatozoa were treated with 0.04% Triton X-100 for extracting the plasma membrane and the suspension was separated into the Triton-soluble and insoluble fractions by centrifugation, only the hydrolytic activity towards succinyl (Suc)-Leu-Leu-Val-Tyr-4-methylcoumaryl-7-amide (MCA), a typical substrate for chymotrypsin-like protease, was mostly retained in the insoluble fraction. The bulk of the activities toward other substrates was detected in the soluble fraction. Flagellar axonemes isolated from demembranated sperm showed considerable hydrolytic activity toward Suc-Leu-Leu-Val-Tyr-MCA and the activity was still retained in the axoneme even after further washing. The hydrolysis was activated by a low concentration of SDS, suggesting that the protease associated with the axonemes is a multicatalytic ATP-dependent proteinase (proteasome). Motility of demembranated sperm was inhibited by Suc-Leu-Leu-Val-Tyr-MCA in an ATP-concentration-dependent manner. These results suggest that proteasomes associated with flagellar axoneme regulate flagellar motility.

Topics: Amino Acid Sequence; Animals; Cell Fractionation; Cell Membrane; Chymotrypsin; Coumarins; Hydrolysis; Male; Molecular Sequence Data; Oligopeptides; Salmon; Sodium Dodecyl Sulfate; Sperm Motility; Sperm Tail; Spermatozoa

To yield biologically active hormones, prohomones are processed by cleavages at paired basic amino acid residues. In this study we report that a chymotrypsin-like activity has been colocalized with trypsin-like and carboxypeptidase-B-like proteases in neurosecretory granules, the site of intracellular processing of prohomones. Using a peptide of 11 amino acids as a simple model system for the study of prohomone processing, we have identified in neurosecretory granule membranes a novel protease that specifically recognizes and cleaves peptide bonds at aromatic residues. Studies were also performed with the fluorogenic peptide substrate N-succinyl-leucyl-leucyl-valyl-tyrosine-7-amino-4-methyl-coumarine . The identified protease activity is inactivated by a cloromethyl ketone derivative (Tos-Phe-CH2-Cl) by Trasylol, and markedly by phenylmethylsulfonylfluoride and diisopropylfluorophosphate. This activity is colocalized with other prohomone-processing enzymes in neurosecretory granules, which indicates that this activity is involved in the prohomone processing. Alternatively, this activity may function in the degradation of homones and neuropeptides when they are released in synapses.

Topics: Amino Acid Sequence; Animals; Carboxypeptidase B; Carboxypeptidases; Cell Fractionation; Centrifugation, Density Gradient; Chymotrypsin; Coumarins; Cytoplasmic Granules; Hormones; Hypothalamus; Intracellular Membranes; Kinetics; Male; Molecular Sequence Data; Oligopeptides; Protease Inhibitors; Protein Precursors; Rats; Rats, Inbred Strains; Trypsin