Enzymatic properties of the proteasome purified from starfish oocytes and its catalytic subunits involved in oocyte maturation

The 20S proteasome was purified from oocytes of the starfish Asterina pectinifera and its enzymatic properties were investigated. The chymotrypsin-like activities were potently inhibited by PSI as well as MG115, whereas the trypsin-like and peptidyl-glutamyl peptide-hydrolyzing (PGPH) activities were not or only weakly inhibited by PSI and MG115. The inhibitory ability of MG115 toward germinal vesicle breakdown (GVBD) coincided with those toward the trypsin-like and PGPH activities, and PSI showed no inhibitory effect on GVBD. We have previously reported that the inhibition pattern toward GVBD of peptidyl-argininals, which potently inhibited the proteasomal trypsin-like activity rather than the chymotrypsin-like activity, correlated with the inhibition pattern toward the chymotrypsin-like activity of the proteasome. These results, together with the peptidyl-argininals scarcely inhibiting the PGPH activity at concentrations sufficient for the inhibition toward GVBD, indicate that both the chymotrypsin-like and trypsin-like activities, but not the PGPH activity, of the proteasome are responsible for degradation of the physiological substrate during starfish oocyte maturation. It was also suggested that the inhibition of a single catalytic site of the proteasome is not sufficient for prevention of the proteasomal function.     

RNA interference of proteasome subunit of PSMbeta7 gene restricts proteasome subunit PSMbeta1 and PSMbeta5 mRNA expression and peptidyl-glutamyl peptide-hydrolyzing proteasome activity in neonatal cardiomyocytes

Using small interfering RNA (siRNA) transfection of neonatal cardiomyocytes to inhibit expression of nonproteolytic proteasome beta7 subunit, we observed a significant decrease in beta1 proteolytic subunit mRNA expression. Proteasome peptidyl-glutamyl peptide-hydrolyzing activity decreased to 28% (0.48 +/- 0.2 nM AMC/min) compared to control (1.7 +/- 0.5 nM AMC/min) (P < 0.05). Beta5 Subunit mRNA expression decreased 21 times (P < 0.05) with no changes in its chymotrypsin-like activity. Proteasome trypsin-like activity and activity of another proteolytic enzyme tripeptidyl-peptidase II remained unchanged.     

Proteasome inactivation upon aging and on oxidation-effect of HSP 90

Increases of oxidatively modified protein in the cell have been associated with the aging process. Such an accumulation of damaged protein may be the result of increase in the rate of protein oxidation and/or decrease in the rate of degradation of oxidized protein. The multicatalytic proteinase or proteasome is known to be the major proteolytic system involved in the removal of oxidized protein. We have reported that, after isolation of the 20S proteasome from the liver of young and old male Fischer 344 rat, out of the three peptidase activities (chymotrypsin-like, trypsin-like and peptidyl-glutamyl peptide hydrolase) we assayed with fluorogenic peptides, the peptidyl-glutamyl peptide hydrolase activity was declining with age to a value approximately 50% of that observed for protease purified from young rats. The proteasome was subjected to metal catalyzed oxidation to determine the susceptibility of the different peptidase activities to oxidative inactivation. Both trypsin-like and peptidyl-glutamyl peptide hydrolase activities were found sensitive to oxidation. Treatment of the proteasome with 4-hydroxy-2-nonenal, a major lipid peroxidation product, was also found to inactivate the trypsin-like activity. However, the trypsin-like activity was protected from inactivation by metal catalyzed oxidation in proteasome preparations contaminated with HSP 90, a protein that often copurifies with the proteasome. Upon addition of HSP 90 to pure 20S active proteasome, the trypsin-like activity was protected from inactivation by metal catalyzed oxidation and from inactivation by treatment with 4-hydroxy-2-nonenal. These results suggest a possible intervention of HSP 90 in response to oxidative stress in preventing the inactivation of the proteasome by oxidative damage. Abbreviations: AAF-amc – Ala-Ala-Phe-7-amido-4-methylcoumarin; LSTR-amc – N-t-Boc-Leu-Ser-Thr-Arg-7-amido-4-methylcoumarin; LLE-na – Leu-Leu-Glu-b-naphthylamide; HSP 90: heat shock protein 90, MCP – multicatalytic proteinase or 20S proteasome.     

Evidence for the existence of a non-catalytic modifier site of peptide hydrolysis by the 20 S proteasome

The 20 S proteasome is an endoprotease complex that preferentially cleaves peptides C-terminal of hydrophobic, basic, and acidic residues. Recently, we showed that these specific activities, classified as chymotrypsin-like, trypsin-like, and peptidylglutamyl peptide-hydrolyzing (PGPH) activity, are differently affected by Ritonavir, an inhibitor of human immunodeficiency virus-1 protease. Ritonavir competitively inhibited the chymotrypsin-like activity, whereas the trypsin-like activity was enhanced. Here we demonstrate that the Ritonavir-mediated up-regulation of the trypsin-like activity is not affected by specific active site inhibitors of the chymo-trypsin-like and PGPH activity. Moreover, we show that the mutual regulation of chymotrypsin-like and PGPH activities by their substrates as described previously by a "cyclical bite-chew" model is not affected by selective inhibitors of the respective active sites. These data challenge the bite-chew model and suggest that effectors of proteasome activity can act by binding to non-catalytic sites. Accordingly, we propose a kinetic "two-site modifier" model that assumes that the substrate (or effector) may bind to an active site as well as to a second non-catalytic modifier site. This model appears to be valid as it describes the complex kinetic effects of Ritonavir very well. Since Ritonavir partially inhibits major histocompatibility complex class I restricted antigen presentation, the postulated modifier site may be required to coordinate the active centers of the proteasome for the production of class I peptide ligands.     

Effect of quercetin on the activity of purified 20S and 26S proteasomes and proteasomal activity in isolated cardiomyocytes

Quercetin inhibits in vitro in dose-dependent manner all three peptidase activities in purified 20S proteasome, the inhibitory effect is comparable to that of a specific proteasome inhibitor. The maximum inhibitory effect of quercetin was observed against the chymotrypsin-like activity of 20S proteasome. Similarly, quercetin inhibits the activity of 26S proteasome from proteasomal fraction II (PF II). Determination of proteasome activity in isolated cardiomyocytes has demonstrated 26% inhibition of trypsin-like proteasomal activity (p = 0.03), 63.7% inhibition of chymotrypsin-like activity (p = 0.04), and 34.2% inhibition of peptidyl-glutamyl peptide hydrolase (p = 0.16) activity by quercetin. Quercetin, its water-soluble analogue corvitin, and clastolactacystin-β-lactone, the specific proteasome inhibitor, exert virtually the same effects on cardiomyocytes. At the concentrations of 5 and 10 μM quercetin corvitin caused the decrease in number of living cardiomyocytes and the increase in number of necrotic and apoptotic cells. At the concentration of 2.5 μM quercetin and corvitin reduced substantially the damaging effect of anoxia-reoxygenation on cardiomyocytes and resulted in decrease in number of necrotic and apoptotic cells. The data obtained suggest that mechanisms of the quercetin cardioprotective effect may involve the inhibition of proteasome activity.     

Participation of 650-kDa protease (20 S proteasome) in starfish oocyte maturation.

A protease involved in oocyte maturation of a starfish, Asterina pectinifera, was explored. Trypsin-like and chymotrypsin-like activities of the 650-kDa protease in oocyte extract were revealed to increase more than twice under the influence of 1-methyladenine before germinal vesicle breakdown (GVBD) during maturation. The inhibitory potencies of leupeptin and its five analogs against the chymotrypsin-like activity, but not the trypsin-like activity, of this protease was well in accord with those against GVBD (Takagi Sawada et al. (1989). Dev. Biol. 133, 609-612). These results indicate that the chymotrypsin-like activity of the 650-kDa protease (most probably 20 S proteasome) plays a key role in starfish oocyte maturation.     

Synthesis and activity of tyropeptin A derivatives as potent and selective inhibitors of mammalian 20S proteasome

Tyropeptin A, a potent proteasome inhibitor, was isolated from the culture broth of Kitasatospora sp. MK993-dF2. We synthesized the derivatives of tyropeptin A to enhance its inhibitory potency. Among the synthesized derivatives, the most potent compound, TP-104, exhibited a 20-fold inhibitory potency enhancement for chymotrypsin-like activity of 20S proteasome compared to tyropeptin A. Additionally, TP-110 specifically inhibited the chymotrypsin-like activity, but did not inhibit the post-glutamyl-peptide hydrolyzing (PGPH) and the trypsin-like activities of 20S proteasome. In vitro TP-110 strongly inhibited the growth of various cell lines.     

Cell-line-specific high background in the Proteasome-Glo assay of proteasome trypsin-like activity

Proteasome-Glo is a homogeneous cell-based assay of proteasomal chymotrypsin-like, trypsin-like, and caspase-like activities using luminogenic substrates, commercially available from Promega. Here we report that the background activity from cleavage of the substrate of the trypsin-like sites by nonproteasomal proteases in multiple breast and lung cancer cell lines exceeds the activity of the proteasome. We also observed substantial background chymotrypsin-like activity in some cell lines. Thus, Proteasome-Glo assay must be used with caution, and it is necessary to include a specific proteasome inhibitor to determine the background for each proteasome activity.     

Peroxynitrite-induced oxidation and its effects on isolated proteasomal systems

The proteasomes are the major intracellular proteolytic systems involved in the removal of altered proteins. In this study, we examined different susceptibilities of constitutive (XYZ) and interferon-gamma inducible (LMP) 20S proteasomes, isolated from bovine brain and thymus, respectively, to peroxynitrite-mediated oxidation. Exposure of XYZ and LMP proteasomes to increasing amounts of peroxynitrite resulted in different levels, in the two enzymes, of 3-nitrotyrosine groups and tryptophan residues oxidation. 1-Anilino-8-naphtalene-sulfonic acid binding studies and quenching of tryptophan residues indicated that the LMP complex was more sensitive to peroxynitrite. Regarding the proteolytic activities, the XYZ proteasome showed an overall activation (even if the trypsin-like (T-L) component was 20% inhibited), with the peptidyl-glutamyl peptide-hydrolyzing (PGPH) and branched-chain amino acid-preferring (BrAAP) activities being the most stimulated. On the other end, the LMP proteasome was inhibited, especially the BrAAP activity, whereas the T-L activity was not affected. Furthermore, exposure to increasing amounts of peroxynitrite induced a gradual decrease of beta-casein degrading rate by the LMP proteasome, whereas it did not influence the constitutive complex. Our results indicated that peroxynitrite caused a mild modification of the XYZ complex, leading to activation of its catalytic activities. Differently, the LMP proteasome showed a more significant conformational change resulting in the inhibition of the proteolytic functions.     

Proteasomal activities in the claw muscle tissue of European lobster, Homarus gammarus, during larval development

Decapod crustaceans grow discontinuously and gain size through complex molt processes. The molt comprises the loss of the old cuticle and, moreover, substantial reduction and re-organization of muscles and connective tissues. In adult lobsters, the muscle tissue of the massive claws undergoes significant atrophy of 40-75% before ecdysis. The degradation of this tissue is facilitated by calcium-dependent proteases and by the proteasome, an intra-cellular proteolytic multi-enzyme complex. In contrast to the adults, the involvement of the proteasome during the larval development is yet not validated. Therefore, we developed micro-methods to measure the 20S and the 26S proteasomal activities within mg- and sub-mg-quantities of the larval claw tissue of the European lobster, Homarus gammarus. Within the three larval stages (Z1-3) we distinguished between sub-stages of freshly molted/hatched (post-molt), inter-molt, and ready to molt (pre-molt) larvae. Juveniles were analyzed in the post-molt and in the inter-molt stage. The trypsin-like, the chymotrypsin-like, and the peptidyl-glutamyl peptide hydrolase activity (PGPH) of the 20S proteasome increased distinctly from freshly hatched larvae to pre-molt Z1. During the Z2 stage, the activities were highest in the post-molt animals, decreased in the inter-molt animals and increased again in the pre-molt animals. A similar but less distinct trend was evident in the Z3 stages. In the juveniles, the proteasomal activities decreased toward the lowest values. A similar pattern was present for the chymotrypsin-like activity of the 26S proteasome. The results show that the proteasome plays a significant role during the larval development of lobsters. This is not only reflected by the elevated activities, but also by the continuous change of the trypsin/chymotrypsin-ratio which may indicate a shift in the subunit composition of the proteasome and, thus, a biochemical adjustment to better cope with elevated protein turnover rates during larval development.     

Structure-based design of derivatives of tyropeptin A as the potent and selective inhibitors of mammalian 20S proteasome

Tyropeptin A, a new potent proteasome inhibitor, was produced by Kitasatospora sp. MK993-dF2. To enhance the inhibitory potency of tyropeptin A, we constructed the structural model of tyropeptin A bound to the site responsible for the chymotrypsin-like activity of mammalian 20S proteasome. Based on these modeling experiments, we designed and synthesized several derivatives of tyropeptin A. Among them, the most potent compound, TP-104, exhibited a 20-fold enhancement in its inhibitory potency compared to tyropeptin A. Additionally, TP-110 specifically inhibited the chymotrypsin-like activity, but did not inhibit the PGPH and the trypsin-like activities.     

Changes in the expression and the enzymic properties of the 20S proteasome in sugar-starved maize roots. evidence for an in vivo oxidation of the proteasome

The 20S proteasome (multicatalytic proteinase) was purified from maize (Zea mays L. cv DEA 1992) roots through a five-step procedure. After biochemical characterization, it was shown to be similar to most eukaryotic proteasomes. We investigated the involvement of the 20S proteasome in the response to carbon starvation in excised maize root tips. Using polyclonal antibodies, we showed that the amount of proteasome increased in 24-h-carbon-starved root tips compared with freshly excised tips, whereas the mRNA levels of alpha 3 and beta 6 subunits of 20S proteasome decreased. Moreover, in carbon-starved tissues, chymotrypsin-like and caseinolytic activities of the 20S proteasome were found to increase, whereas trypsin-like activities decreased. The measurement of specific activities and kinetic parameters of 20S proteasome purified from 24-h-starved root tips suggested that it was subjected to posttranslational modifications. Using dinitrophenylhydrazine, a carbonyl-specific reagent, we observed an increase in carbonyl residues in 20S proteasome purified from starved root tips. This means that 20S proteasome was oxidized during starvation treatment. Moreover, an in vitro mild oxidative treatment of 20S proteasome from non-starved material resulted in the activation of chymotrypsin-like, peptidyl-glutamyl-peptide hydrolase and caseinolytic-specific activities and in the inhibition of trypsin-like specific activities, similar to that observed for proteasome from starved root tips. Our results provide the first evidence, to our knowledge, for an in vivo carbonylation of the 20S proteasome. They suggest that sugar deprivation induces an oxidative stress, and that oxidized 20S proteasome could be associated to the degradation of oxidatively damaged proteins in carbon starvation situations.     

Lovastatin and simvastatin are modulators of the proteasome

Lovastatin and simvastatin are HMG-CoA reductase inhibitors widely used as antihyperlipidemic drugs, which also display antiproliferative properties. In the present paper, we provide evidence that both lovastatin and simvastatin are modulators of the purified bovine pituitary 20 S proteasome, since they mildly stimulate the chymotrypsin-like activity and inhibit the peptidylglutamylpeptide hydrolyzing activity without interfering with the trypsin-like activity. However, those effects are only observed when the closed ring forms of the drugs are used, while the opened ring form of lovastatin acts as a mild inhibitor of the chymotrypsin like activity. The closed ring form of lovastatin is much more potent as a cytotoxic agent on the Colon-26 (C-26) colon carcinoma cell line than the opened ring form, which is only mildly cytostatic. Moreover, neither the cytotoxic effects nor the effects on 20 S proteasome activities are prevented by mevalonate, which by itself inhibits the trypsin-like activity of the proteasome. Neither the opened ring nor the closed ring form of lovastatin induces an accumulation of ubiquitin-protein conjugates, which is observed after treatment with lactacystin, a selective proteasome inhibitor. In contrast with the opened ring form of lovastatin, the closed ring form induces the disappearance of detectable p27(kip1) from C-26 cells. Altogether, our results indicate that the closed ring form of lovastatin induces cytotoxic effects independent of its HMG-CoA inhibiting activity, however, those effects are mediated by a complex modulation of proteasome activity rather than by inhibition of the 20 S proteasome.     

Regulation of the 26S proteasome activities by peptides mimicking cleavage products

The implication of the released peptides in allosteric effects during protein degradation catalyzed by the proteasome is an important question not completely resolved. We present here data showing modulation of 26S proteasome activities by peptides composed of 5 or 6 natural amino acids that mimic the products generated during protein breakdown. Several of these peptides inhibit the chymotrypsin-like activity of the Xenope 26S proteasome whereas its trypsin-like activity is enhanced. The basic peptides produced competitive inhibition of the chymotrypsin-like activity and the acidic peptides, parabolic inhibition involving two different binding sites. Our results are in agreement with a model involving hypothetical non-catalytic sites interacting with effectors to modulate the peptidase activities of the proteasome. They also suggest that allosteric effects may occur in the proteasome during protein degradation.     

The caspase-like sites of proteasomes,their substrate specificity,new inhibitors and substrates,and allosteric interactions with the trypsin-like sites

Proteasomes are the primary sites for protein degradation in mammalian cells. Each proteasome particle contains two chymotrypsin-like, two trypsin-like, and two caspase-like proteolytic sites. Previous studies suggest a complex network of allosteric interactions between these catalytic and multiple regulatory sites. We used positional scanning combinatorial substrate libraries to determine the extended substrate specificity of the caspase-like sites. Based on this analysis, several new substrates were synthesized, the use of which confirmed earlier observations that caspase-like sites (often termed postglutamyl peptide hydrolase) cleave after aspartates better than after glutamates. Highly selective inhibitors of the caspase-like sites were also generated. They stimulated trypsin-like activity of yeast 20 S proteasomes up to 3-fold but not when binding of the inhibitor to the caspase-like sites was prevented in a mutant carrying an uncleaved propeptide. Although substrates of the caspase-like sites allosterically inhibit the chymotrypsin-like activity, inhibitors of the caspase-like sites do not affect the chymotrypsin-like sites. Furthermore, when caspase-like sites were occupied by the uncleaved propeptide or inhibitor, their substrates still inhibited the chymotrypsin-like activity. Thus, occupancy of the caspase-like sites stimulates the trypsin-like activity of proteasomes, but substrates of the caspase-like sites inhibit the chymotrypsin-like activity by binding to a distinct noncatalytic site.     

Novel beta-lactam derivatives: potent and selective inhibitors of the chymotrypsin-like activity of the human 20S proteasome

A series of beta-lactam derivatives has been designed and synthesized to inhibit the chymotrypsin-like activity of the human 20S proteasome. The most potent compounds of this new structural class of beta-subunit selective 20S proteasome inhibitors exhibit IC50 values in the low-nanomolar range and show good selectivity over the trypsin-like and post-glutamyl-peptide hydrolytic activities of the enzyme.     

Increased expression and altered subunit composition of proteasomes induced by continuous proteasome inhibition establish apoptosis resistance and hyperproliferation of Burkitt lymphoma cells

The proteasome is the main protease for extralysosomal protein degradation in eukaryotic cells, and constitutes a sophisticated high molecular mass proteinase complex underlying a tightly coordinated expression and assembly of multiple subunits and subcomplexes. Here we show that continuous inhibition of proteasomal chymotrypsin-like peptidase activity by the proteasome inhibitor bortezomib induces in human Namalwa Burkitt lymphoma cells increased de novo biogenesis of proteasomes accompanied by increased expression of the proteasome maturation protein POMP, increased expression of 19S-20S-19S proteasomes, and abrogation of expression of beta 1i, beta 2i and beta 5i immunosubunits and PA28 in favor of increased expression of constitutive proteolytic beta1, beta2 and beta 5 subunits and 19S regulatory complexes. These alterations of proteasome expression and subunit composition are accompanied by an increase in proteasomal caspase-like, trypsin-like and chymotrypsin-like peptidase activities, not inhibitable by high doses of bortezomib. Cells harboring these proteasomal alterations display rapid proliferation and cell cycle progression, and acquire resistance to apoptosis induced by proteasome inhibitors, gamma-irradiation and staurosporine. This acquired apoptosis resistance is accompanied by de novo expression of anti-apoptotic Hsp27 protein and the loss of ability to accumulate and stabilize pro-apoptotic p53 protein. Thus, increased expression, altered subunit composition and increased activity of proteasomes constitute a hitherto unknown adaptive and autoregulatory feedback mechanism to allow cells to survive the lethal challenge of proteasome inhibition and to establish a hyperproliferative and apoptosis-resistant phenotype.     

Structure-based optimisation of 2-aminobenzylstatine derivatives: potent and selective inhibitors of the chymotrypsin-like activity of the human 20S proteasome

We have identified 2-aminobenzylstatine derivatives that inhibit non-covalently the chymotrypsin-like activity of the human 20S proteasome. A structure-based optimisation approach has allowed us to improve the potency of this structural class of proteasome inhibitors from micromolar to nanomolar level. The new derivatives showed good selectivity against the trypsin-like and post-glutamyl-peptide hydrolytic activities of this enzyme.     

Enzymatic properties of the 20S proteasome in wheat endosperm and its biochemical characteristics after seed imbibition

The 20S proteasome from wheat ( Triticum aestivum L., Yangmai 158) endosperm was purified to apparent homogeneity by three sequential centrifugations and gradient PAGE (GPAGE). The purified 20S proteasome clearly cleaved peptidyl-arylamide bonds in the model synthetic substrates Z-GGL-AMC and Z-GGR-AMC, which are used to reflect chymotrypsin-like and trypsin-like activity, respectively. For both substrates, the optimum pH was 8.0, but the optimum temperatures for chymotrypsin-like and trypsin-like activity were 55 °C and 37 °C, respectively. Both enzyme activities were clearly inhibited by MG115 and PMSF. Polyubiquitinated proteins remained constant from 0 to 7 days after seed imbibition, but caseinolytic activity and the amount of the 20S proteasome associated with the aleurone layer decreased from 1 to 2 days after imbibition (DAI), then increased from 2 to 4 DAI, and reached a maximum at 4 DAI that was retained until 7 DAI. An increase was seen in the mRNA level of the β5 subunit of the 20S proteasome from 2 DAI, and caseinolytic activity and the amount of the 20S proteasome increased from 3 DAI onwards. In addition, the main storage proteins of the wheat endosperm could not be hydrolyzed by the 20S proteasome. The evidence suggests that the main role of the 20S proteasome may not be to degrade massive proteins of the wheat endosperm after seed imbibition.     

Crude and purified proteasome activity assays are affected by type of microplate

Measurement of proteasome activity is fast becoming a commonly used assay in many laboratories. The most common method to measure proteasome activity involves measuring the release of fluorescent tags from peptide substrates in black microplates. Comparisons of black plates used for measuring fluorescence with different properties show that the microplate properties significantly affect the measured activities of the proteasome. The microplate that gave the highest reading of trypsin-like activity of the purified 20S proteasome gave the lowest reading of chymotrypsin-like activity of the 20S proteasome. Plates with medium binding surfaces from two different companies showed an approximately 2-fold difference in caspase-like activity for purified 20S proteasomes. Even standard curves generated using free 7-amino-4-methylcoumarin (AMC) were affected by the microplate used. As such, significantly different proteasome activities, as measured in nmol AMC released/mg/min, were obtained for purified 20S proteasomes as well as crude heart and liver samples when using different microplates. The naturally occurring molecule betulinic acid activated the chymotrypsin-like proteasome activity in three different plates but did not affect the proteasome activity in the nonbinding surface microplate. These findings suggest that the type of proteasome activity being measured and sample type are important when selecting a microplate.