Trypsin-like Activity Of Proteasome Research Articles

Evaluation of the Anti-Trypanosomal Activity of Tyropeptin A

The natural compound tyropeptin A, a new peptidyl aldehyde proteasome inhibitor, was tested for its trypanocidal activity in vitro using culture-adapted bloodstream forms of Trypanosoma brucei. The concentrations of tyropeptin A required to reduce the growth rate by 50 % and to kill all cells were 10 and 100 times lower for bloodstream-form trypanosomes than for human leukaemia HL-60 cells, respectively. Enzymatic analysis showed that the trypsin-like activity of the trypanosome proteasome and the chymotrypsin-like activity of the mammalian proteasome are particularly sensitive to inhibition by tyropeptin A. The results suggest that natural compounds targeting the trypsin-like activity of the proteasome may serve as leads for rational drug development of novel anti-trypanosomal agents.

Renal cytoplasmic proteasome proteinase activities are altered in chronic renal failure

The effect of uremia on renal cortex cytoplasmic proteasomes was examined by comparing proteasomes isolated from 5/6th nephrectomy rats 3-months post-surgery and age-matched control rats with normal renal function. ATP-dependent proteasome activity was reduced 50% in chronic renal failure rats (CRF) 3-months post-surgery compared to age-matched control rats. Trypsin-like (T-like) proteasome activity was decreased 90% compared to 70% for caspase-like activity (PGPHase) and 30% for chymotrypsin-like activity (C-like). ATP-independent proteasome activity was decreased 60% in CRF rats 3-months post-surgery. ATP-independent renal cortex proteasome T-like activity in CRF rats was 4% of age-matched control rats. C-like and PGPHase activities were 60% and 50% of age-matched controls, respectively. Uremia was associated with decreased 26S proteasome β subunits. CRF rat 26S proteasomes had decreased levels of β1, β3, α4, and α7 abundances. Compared to age-matched control rats with normal renal function, CRF rats had a 25% increase in ubiquitinated cytoplasmic proteins. Decreased renal cytoplasmic proteasome activity may play a role in renal tubule hypertrophy common to renal diseases associated with decreased functioning nephrons.

317 PUBLICATION Epidermal growth factor receptor levels in progesterone receptor positive breast tumors

Parkin suppression induces accumulation of β-amyloid in mutant tau mice. We studied the effect of parkin suppression on behaviour and brain pathology in APPswe mutant mice. We produced double mutant mice with human mutated APPswe + partial (hemizygote) or total (homozygote) deletion of Park-2 gene. We studied the development, behaviour, brain histology, and biochemistry of 12- and 16-month-old animals in 6 groups of mice, with identical genetic background: wild-type (WT), APPswe overexpressing (APP), hemizygote and homozygote deletion of Park-2 (PK+/− and PK−/−, respectively), and double mutants (APP/PK+/− and APP/PK−/−).APP mice have reduced weight gain, decreased motor activity, and reduced number of entrances and of arm alternation in the Y-maze, abnormalities which were partially or completely normalized in APP/PK+/− and APP/PK−/− mice. The double mutants had similar number of mutant human APP transgene copies than the APP and levels of 40 and 80 kDa proteins; but both of them, APP/PK+/− and APP/PK−/− mice, had less plaques in cortex and hippocampus than the APP mice. APP mutant mice had increased apoptosis, proapoptotic Bax/Bcl2 ratios, and gliosis, but these death-promoting factors were normalized in APP/PK+/− and APP/PK−/− mice. APP mutant mice had an increased number of tau immunoreactive neuritic plaques in the cerebral cortex as well as increased levels of total and phosphorylated tau protein, and these changes were partially normalized in APP/PK+/− heterozygotic and homozygotic APP/PK−/− mice. Compensatory protein-degrading systems such as HSP70, CHIP, and macroautophagy were increased in APP/PK+/− and APP/PK−/−. Furthermore, the chymotrypsin- and trypsin-like proteasome activities, decreased in APP mice in comparison with WT, were normalized in the APP/PK−/− mice.We proposed that partial and total suppression of parkin triggers compensatory mechanisms, such as chaperone overexpression and increased autophagy, which improved the behavioural and cellular phenotype of APPswe mice.

Peptidyl Vinyl Ester Derivatives: New Class of Selective Inhibitors of Proteasome Trypsin-Like Activity

The proteasome is a multicatalytic proteinase complex which plays a central role in intracellular protein degradation. We report here the synthesis and biological activities of a new class of specific proteasome inhibitors selective for trypsin-like activity. These tripeptide-based compounds bearing a C-terminal vinyl ester are nontoxic, and do not affect cell proliferation, but are able to modulate the generation and presentation of immunogenic peptides presented by MHC class I molecules.

Immunoproteasome and LMP2 polymorphism in aged and Alzheimer's disease brains

In this study, we investigated the presence and role of immunoproteasome and its LMP2 subunit polymorphism at codon 60 in Alzheimer's disease (AD). Immunoproteasome was present in brain areas such as hippocampus and cerebellum and localized in neurons, astrocytes and endothelial cells. A higher expression of immunoproteasome was found in brain of AD patients than in brain of non-demented elderly, being its expression in young brain negligible or absent. Furthermore, AD affected regions showed a partial decrease in proteasome trypsin-like activity. The study of LMP2 polymorphism (R/H) showed that it does not influence LMP2 expression (neither the mRNA nor mature protein) in brain tissue. However, control brain areas of AD patients carrying the RR genotype showed an increased proteasome activity in comparison with RH carriers. To test whether this effect of the genotype might be related to AD onset we performed a genetic study, which allowed us to exclude an association of LMP2 codon 60 polymorphism with AD onset, despite its influence on the proteasome activity in human brain.

Life long calorie restriction increases heat shock proteins and proteasome activity in soleus muscles of Fisher 344 rats

Heat shock proteins (HSP's) closely interact with 20S proteasome and have been shown to maintain catalytic activity, responsible for the prevention of protein aggregation. A decrease in both proteasome activity and heat shock proteins (HSP's) has been observed with age. We investigated whether life-long calorie restriction (CR), a natural intervention, which prolongs life span, could prevent the age-associated decline in HSP's and restore the proteolytic activity of the 20S proteasome in skeletal muscle. Hence, we investigated HSP's and proteasome activity in the soleus muscle from 12-mo-old (Adult) and 26–28mo old ad libitum fed (Old), and 26–28mo old CR (Old-CR; fed 40% of ad libitum for their lifespan) male Fisher 344 rats. Trypsin-like proteasome activity in Old rats was significantly less than both Adult and Old-CR rats. Furthermore, no significant changes where found in chymotrypsin-like proteasome activity due to age or diet. Levels of HSP 72 and 25 were significantly less in Old animals when compared to both Adult and Old-CR rats. In contrast, HSP 90 was elevated in Old rats by 220% compared to adult animals and life-long calorie restriction caused a significant induction (150%) compared to age-matched ad libitum fed animals. Protein carbonyls were significantly elevated in Old when compared to Adult rats, but showed no significant decline due to life long CR. This study shows that HSP's may be largely responsible for the restoration of the trypsin-like activity of the 20S proteasome with age. The large increase in HSP 90 is intriguing and further studies are required to elucidate its role in maintaining 20S proteasome function.

Trypanocidal effect of α′,β′-epoxyketones indicates that trypanosomes are particularly sensitive to inhibitors of proteasome trypsin-like activity

Previous studies have shown that the proteasome of Trypanosoma brucei is a candidate for novel chemotherapy of African sleeping sickness. In this study, two potent and highly selective α′,β′-epoxyketones peptide proteasome inhibitors, epoxomicin and YU101, have been tested for their trypanocidal activities in vitro using culture-adapted bloodstream forms of T. brucei. Both inhibitors displayed promising anti-trypanosomal activities with ED 50 and ED 90 values in the low to mid nanomolar range. Based on MIC values, epoxomicin exhibited a selectivity index approaching those of commercially available drugs. Enzymatic analyses of proteasomal peptidase activities revealed that, compared with mammalian cells, trypanosomes are particular sensitive to inhibition of the trypsin-like activity of the proteasome. In conclusion, the data suggests that proteasome inhibitors targeting the trypsin-like activity are the rational choice for future anti-trypanosomal drug development.

Nitric oxide inhibits H2O2-induced transferrin receptor-dependent apoptosis in endothelial cells: Role of ubiquitin-proteasome pathway.

We investigated here the mechanism of cytoprotection of nitric oxide (*NO) in bovine aortic endothelial cells treated with H2O2. NONOates were used as *NO donors that released *NO slowly at a well defined rate in the extracellular and intracellular milieus. H2O2-mediated intracellular dichlorofluorescein fluorescence and apoptosis were enhanced by the transferrin receptor (TfR)-mediated iron uptake. *NO inhibited the TfR-mediated iron uptake, dichlorofluorescein fluorescence, and apoptosis in H2O2-treated cells. *NO increased the proteasomal activity and degradation of nitrated TfR via ubiquitination. Nomega-nitro-L-arginine methyl ester, a nonspecific inhibitor of endogenous *NO biosynthesis, decreased the trypsin-like activity of 26S proteasome. *NO, by activating proteolysis, mitigates TfR-dependent iron uptake, dichlorodihydrofluorescein oxidation, and apoptosis in H2O2-treated bovine aortic endothelial cells. The relevance of biological nitration on redox signaling is discussed.

Glutathiolation of the Proteasome Is Enhanced by Proteolytic Inhibitors

The proteasome inhibitors lactacystin, clastro lactacystin β-lactone, or tri-leucine vinyl sulfone (NLVS), in the presence of [35S]cysteine/methionine, caused increased incorporation of 35S into cellular proteins, even when protein synthesis was inhibited by cycloheximide. This effect was blocked by incubation with the glutathione synthesis inhibitor buthionine sulfoximine. Proteasome inhibitors also enhanced total glutathione levels, increased reduced/oxidized glutathione ratio (GSH/GSSG) and upregulated γ-glutamylcysteine synthetase (rate-limiting in glutathione synthesis). Micromolar concentrations of GSH, GSSG, or cysteine stimulated the chymotrypsin-like activity of purified 20S proteasome, but millimolar GSH or GSSG was inhibitory. Interestingly, GSH did not affect 20S proteasome's trypsin-like activity. Enhanced proteasome glutathiolation was verified when purified preparations of the 20S core enzyme complex were incubated with [35S]GSH after pre-incubation with any of the inhibitors. NLVS, lactacystin or clastro lactacystin β-lactone may promote structural modification of the 20S core proteasome, with increased exposure of cysteine residues, which are prone to S-thiolation. Three main conclusions can be drawn from the present work. First, proteasome inhibitors alter cellular glutathione metabolism. Second, proteasome glutathiolation is enhanced by inhibitors but still occurs in their absence, at physiological GSH and GSSG levels. Third, proteasome glutathiolation seems to be a previously unknown mechanism of proteasome regulation in vivo.

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.

Mutational analysis of subunit iβ2 (MECL-1) demonstrates conservation of cleavage specificity between yeast and mammalian proteasomes

Proteasomes are the major protein-degrading complexes in the cytosol and regulate many cellular processes. To examine the functional importance of the MC14/MECL-1 proteasome active site subunits, cell lines expressing a catalytically inactive form of MECL-1 were established. Whereas mutant MECL-1 was readily incorporated into cytosolic proteasomes, replacing the constitutive MC14 subunit, removal of the prosequence was incomplete indicating that its processing required autocatalytic cleavage. Functional analyses showed that the absence of the MC14/MECL-1 active sites abrogated proteasomal trypsin-like activity, but did not affect other catalytic activities. Our data demonstrate a conservation of cleavage specificity between mammalian and yeast proteasomes.

TMC-86A, B and TMC-96, new proteasome inhibitors from Streptomyces sp. TC 1084 and Saccharothrix sp. TC 1094. I. Taxonomy, fermentation, isolation, and biological activities.

TMC-86A, B and TMC-96, new 20S proteasome inhibitors with an epoxy-beta-aminoketone moiety, were isolated from the fermentation broth of Streptomyces sp. TC 1084 and Saccharothrix sp. TC 1094, respectively. TMC-86A, B and TMC-96 inhibited the chymotrypsin-like and peptidylglutamyl-peptide hydrolyzing activities of 20S proteasome with the following IC50 values: TMC-86A, 5.1 microM and 3.7microM; TMC-86B, 1.1 microM and 31 microM; TMC-96, 2.9 microM and 3.5 microM, respectively. TMC-86A, B and TMC-96 exhibited the weak inhibitory activity against the trypsin-like activity of 20S proteasome with IC50 values of 51 microM, 250 microM, and 36 microM, respectively. They did not inhibit m-calpain, cathepsin L, and trypsin at 100 microM, suggesting their high specificity for proteasome. Taxonomy of the producing strains is also described.

Irreversible potent activation and reversible inhibition of trypsin-like activity of 20S proteasome purified from Xenopus oocytes by fatty acid.

The 20S proteasome purified from animal cells has various latent peptidase activities. Fatty acids such as linoleic, linolenic and oleic acids strongly activate both the chymotrypsin-type and peptidylglutamylpeptide (PGP) hydrolase-type activities, but have been reported to have little activation or inhibition of the trypsin-type activity. We show here that an increase of the fatty acid concentration produces activation of chymotrypsin-type and PGP hydrolase-type in a biphasic fashion: no effect until the threshold concentration and then a sharp activation. In contrast, the trypsin-type activity was markedly inhibited at low concentrations of fatty acid, slightly activated at higher concentrations, and inhibited again at even higher concentrations. The inhibition was removed when the concentration of fatty acid was reduced by dilution after pre-incubation with the fatty acid. As a result, the activation pattern became biphasic, which was identical to that of chymotrypsin-type and PGP hydrolase-type activities. These results suggest that in the chymotrypsin-type and PGP hydrolase-type peptidase fatty acids bind first to a class of sites without direct effect on the peptidase activity, but after saturation of this class it permits more fatty acid to bind to another class of sites involved in the activation. In the trypsin-type peptidase an additional class of fatty acid binding sites is uniquely present, which is involved in the enzyme inhibition. The dilution procedure described above removes the fatty acid molecules bound to the inhibition sites, but not the fatty acid molecules bound to the activation sites; this results in the fatty acid activation profile indistinguishable from that of the chymotrypsin- and PGP hydrolase-type peptidases.

Identification of a 10 S Trypsin-like Protease That Cross-Reacts with Anti-Proteasome Antibody in Sea Urchin Egg Jelly

We have identified 27- and 26-kDa polypeptides in sea urchin egg jelly, both of which cross-reacted with the antibody against 20 S proteasome (multicatalytic proteinase) isolated from sea urchin sperm. Separation of egg jelly fraction by gel filtration or sucrose density gradient centrifugation revealed that these polypeptides comigrated as a complex with a molecular size much smaller than that of proteasome: the apparent molecular mass and the sedimentation coefficient were 200 kDa and 10 S, respectively. This protease significantly hydrolyzed the fluorogenic synthetic substrates for trypsin-like protease but little hydrolyzed those for chymotrypsin-like protease. Trypsin-like activity of sperm proteasome was activated up to more than threefold by a low concentration of sodium dodecyl sulfate (SDS), whereas the egg jelly 10 S protease was inhibited by SDS. Two-dimensional immunoblot and peptide mapping revealed that the 26-kDa polypeptide is a degradative product of 27-kDa polypeptide and that the 10 S protease is composed of a proteasome-related single 27-kDa polypeptide and its modified forms. These results indicate the presence of a 10 S novel assembly of a proteasome subunit only with trypsin-like activity.

Identification and localization of a cysteinyl residue critical for the trypsin-like catalytic activity of the proteasome.

Chemical modification of the proteasome with N-ethylmaleimide (NEM) was performed for the purpose of identifying amino acid residues that play a role in the enzyme's proteolytic function. Modification of the proteasome with NEM specifically and irreversibly suppressed one of the three peptidase activities of the enzyme, viz., the "trypsin-like" activity. Leupeptin, a reversible competitive inhibitor of this activity, protected the activity from NEM inactivation, suggesting that NEM modifies a residue in the leupeptin binding site. Comparisons of enzyme samples labeled with [14C]NEM either in the presence or in the absence of leupeptin allowed the identification of a proteasome subunit containing an NEM-modified, leupeptin-protected cysteinyl residue. The leupeptin protection experiments suggest that residues of this subunit contribute to the active site responsible for the proteasome's trypsin-like activity. This subunit was purified by reverse-phase high-performance liquid chromatography. Peptide mapping and N-terminal amino acid sequencing were employed to acquire information about the primary structure of the subunit, including the sequence surrounding the leupeptin-protected cysteinyl residue. The sequencing data suggest that this proteasome subunit is evolutionarily related to other proteasome subunits that have been sequenced, which show no homology to other known proteases. The assignment of a catalytic function to a member of the proteasome family supports the hypothesis that proteasome subunits represent a structurally and possibly mechanistically novel group of proteases.