Peptidylglutamyl-peptide Hydrolase Activity Research Articles

Hydrogen Peroxide-Induced Oxidative Stress Activates Proteasomal Trypsin-Like Activity in Human U373 Glioma Cells.

Degradation of oxidized or oxidatively modified proteins is an essential part of the cellular antioxidant defense system. 4-Hydroxy-2-nonenal, a major reactive aldehyde formed by lipid peroxidation, causes many types of cellular damage. The major proteolytic system for modified protein degradation is the ubiquitin-proteasome pathway. However, our previous studies using U937 human leukemic cells showed that 4-hydroxy-2-nonenal-modified glyceraldehyde-3-phosphate dehydrogenase (GAPDH) is degraded by cathepsin G. In the present study, U373 human glioma cells were cultured in the presence of hydrogen peroxide (H2O2) to investigate the relationships of proteasome and/or cathepsin G activities and H2O2-induced GAPDH degradation. Treatment of cells with H2O2 for 5 h in culture decreased GAPDH activity as well as its protein concentration in a concentration-dependent manner. Two proteasomal activities (peptidylglutamyl-peptide hydrolase and chymotrypsin-like hydrolase activities) and cathepsin G activity were decreased by H2O2 treatment in a concentration-dependent manner, but proteasomal trypsin-like hydrolase activity increased with cell exposure to high H2O2 concentrations. Among the protease inhibitors examined here, H2O2-induced activation of trypsin-like activity and GAPDH degradation were inhibited by the proteasome inhibitor lactacystin. Furthermore, H2O2-induced activation of trypsin-like activity was also inhibited by another proteasome inhibitor MG-132. These results suggested that proteasomal trypsin-like activity played an important role in eliminating oxidatively modified GAPDH formed in these cells during H2O2 exposure.

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.

Effect of a conserved peptide derived from Kunitz domain of hepatitis B virus x protein on the cell cycle and apoptosis of HepG2 cells via the proteasome pathway

Background Hepatitis B virus (HBV) x protein (HBx) in HepG2 cells causes a moderate decrease in proteolysis activity of the proteasome. A highly conserved Kunitz-type serine protease inhibitor domain within 154 amino acid residues of HBx has been identified. In this study, a peptide chain derived from the Kunitz domain (PKD) was used to study its effect on the cell cycle and apoptosis of HepG2 cells, and investigated the function of PKD on the activities of proteasomes and AAA-ATPase p97, which involves in the ubiquitin-proteasome protein degradation pathway. Methods The PKD peptide (Phe-Val-Leu-Gly-Gly-Cys-Arg-His-Lys) was chemically synthesized. MTT assays were used to determine the effects of PKD on HepG2 cell growth. Mouse anti-p97 antibody was developed for Western blotting to detect the expression of p97. ATPase activity of proteasomes was measured using a colorimetric assay. Peptidase activities of proteasomes were analyzed with various peptidase-specific fluorogenic peptide substrates. Flow cytometry was used to determinate cell cycle phase and apoptosis. Results Viability of HepG2 cells decreased in a PKD-dose-dependent manner. Cells exhibited significant cytotoxicity in the presence of 15 mmol/L of PKD. Western blotting analysis showed that expression of p97 was suppressed in HepG2 cells treated with PKD compared to untreated cells. The ATPase activity of proteasomes from immunoprecipitates of HepG2 cells pretreated with PKD was apparently decreased. Chymotryptic activity of proteasomes in HepG2 cells was significantly inhibited by 10mmol/L PKD; tryptic activity and peptidylglutamyl peptide hydrolase activity of proteasomes were less inhibited by PKD than chymotryptic activity. The cell cycle phase of HepG2 cells treated with PKD for 36 hours was blocked largely at the G0-G1 phase, while untreated control cells were mainly in S phase. PKD also significantly induced apoptosis. Conclusions The peptide derived from Kunitz domain of HBx protein induces HepG2 cell growth arrest and apoptosis, which may result from down-regulation of p97 expression, and decrease of both the ATPase and chymotryptic activities of proteasomes.

Sperm-Surface ATP in Boar Spermatozoa is Required for Fertilization: Relevance to Sperm Proteasomal Function

Extracellular ATP has been implicated in a number of cellular events, including mammalian sperm function. The complement of ATP-dependent sperm proteins includes six subunits of the 26S proteasome, a multi-subunit protease specific to ubiquitinated substrate-proteins. Proteolysis of ubiquitinated proteins by the 26S proteasome is necessary for the success of mammalian fertilization, including but not limited to acrosomal exocytosis (AE) and sperm-zona pellucida (ZP) penetration. The 26S proteasome is uniquely present on the sperm acrosomal surface during mammalian, ascidian, and invertebrate fertilization. The proteasome is a multi-subunit protease complex of ∼2 MDa composed of the 19S regulatory complex and a 20S proteolytic core. Integrity of the 19S complex is maintained by six 19S ATPase subunits (PSMC1 through PSMC6). Consequently, we hypothesized that fertilization will be blocked by the depletion of sperm-surface associated ATP (ssATP). Depletion of ssATP by the Solanum tuberosum apyrase, a 49 kDa, non-cell permeant enzyme, significantly reduced the ATP content measured by an adapted luminescence-ATP assay from which all permeabilizing agents were excluded. Addition of active apyrase to porcine in vitro fertilization (IVF) medium caused a concentration dependent reduction in the overall fertilization rate. No such outcomes were observed in control groups using heat-inactivated apyrase. Apyrase treatment altered the band pattern of 19S ATPase subunits PSMC1 (Rpt2) and PSMC4 (Rpt3) in Western blotting, suggesting that it had an effect on the integrity of the sperm proteasomal 19S complex. Apyrase only altered the proteasomal core activities slightly, since these activities are not directly dependent on external ATP. In contrast, sperm treatment with MG132, a specific inhibitor of the proteasomal core chymotrypsin-like activity, inhibited the target proteolytic activity, but also induced a compensatory elevation in proteasomal peptidyl-glutamyl peptide hydrolase activity. Altogether, the present data provide an important missing piece of evidence in support of the ssATP-dependent, proteasomal-proteolytic model of sperm-ZP interactions.

Alterations of activity and intracellular distribution of the 20S proteasome in ageing retinal pigment epithelial cells

Age-related macular degeneration (AMD) remains high incidence and accounts for a main cause of blindness in ageing people, but its mechanism is still poorly understood. Ageing and associated dysfunction of retinal pigment epithelial (RPE) cells were believed to be the pathological onset of AMD. 20S proteasome has been tightly correlated with cell ageing due to its fundamental role in maintaining cellular homeostasis, but its implication in the ageing process of human RPE cells was seldom concerned. This study aimed to demonstrate the interconnections between 20S proteasome and ageing RPE cells by characterizing age-dependent alterations of the 20S proteasome in primarily cultured human RPE cells. For this purpose, a replicative ageing RPE cell model was established and validated through testing the cell viability, β-galactosidase activity and cellular autofluorescence. Decline in chymotrypsin-like, peptidylglutamyl-peptide hydrolase and trypsin-like activities of the 20S proteasome was detected in aged RPE cells through degradation of fluorogenic substrates. Immunofluorescence assay revealed that the 20S proteasome was concentrated in RPE nucleus, and redistributed partly to the peri-nuclear regions in old RPE passages. These age-dependent changes of the 20S complex were accompanied with a significantly increased fluorescent intensity of intracellular oxidized proteins. Further analysis of the proteasome-to-oxidized protein ratio indicated a preferred protection of the RPE nuclear proteins by the 20S proteasome, which also subsided remarkably as a function of the cell ageing. In conclusion, we demonstrated functional impairment and redistribution of the 20S proteasome with age in human RPE cells and supposed these alterations impactful on the process of RPE cell ageing and furthermore on the pathogenesis of AMD. Future researches on the mechanism of these alterations and the pathways to manipulate their effects are still strongly recommended.

Proteasome Activity Deregulation in LEC Rat Hepatitis: Following the Insights of Transcriptomic Analysis

LEC rats show spontaneous hepatitis and hepatocarcinoma development related to oxidative stress due to abnormal copper accumulation in the liver. We used DNA microarrays bearing 22,012 genes to investigate at the transcriptomic level the progression of the hepatitis in LEC rats in comparison to a control obtained from LEC rats treated with D-penicillamine, a copper chelating agent known to block hepatitis development. Multivariate statistical analyses as partial least square (PLS) regression between transcriptomic data and hepatitis markers in plasma led us to select 483 genes related to hepatitis development in these rats. After a complementary discriminant analysis (PLS-DA), 239 important genes for the separation between the different rat groups were selected. Gene ontology classification revealed an overrepresentation of genes involved in protein metabolism-related functions. More importantly, some genes implicated in proteasome pathway were upregulated. However, analysis of 20S proteasome activity showed that trypsin-like and peptidylglutamyl peptide hydrolase activities were diminished during hepatitis. Because oxidative stress is known to promote the inactivation of the proteasome complex, we propose the deregulation of the proteasome genes expression as a result of oxidative inactivation of proteasome activity during hepatitis in LEC rats. These results bring new insights in the hepatitis and the hepatocarcinogenesis development.

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.

Characterization and localization of Plasmodium falciparum homolog of prokaryotic ClpQ/HslV protease

The β subunits (β1, β2, and β5) of 20S proteasome and HslV/ClpQ are ATP-dependent threonine proteases present in eukaryotes and prokaryotes, respectively that control levels of key regulatory proteins in the cell. The orthologue of prokaryotic HslV protease in Plasmodium falciparum (PfHslV) is a novel drug target candidate that has no homolog in the human host. In the present study, the PfHslV was expressed, localized and biochemically characterized. The recombinant PfHslV harbored threonine protease specific activity as well as chymotrypsin like and peptidyl glutamyl peptide hydrolase activities. All the three activities could be inhibited by respective specific inhibitors. The protein was localized in the cytosol of the parasite as a soluble protein by Western immunoblotting of parasite fractions and by immuno-fluorescence microscopy. Activity of the protease in the parasite was ascertained by following the degradation of GFP in a transgenic parasite line expressing fusion protein of GFP and Arc-repressor gene, a known target of HslV protease in the prokaryotes. A model structure of PfHslV was constructed based on the crystal structure of Escherichia coli HslV to assess the structural homology. Availability of the structure model of PfHslV may facilitate identification or designing of novel and specific drugs against PfHslV. The in vitro protease assays with recombinant PfHslV and the transgenic parasite line generated in the present study may be exploited in the screening of novel inhibitors to evaluate their anti-malarial activity.

Down-regulation of estrogen receptor-α in MCF-7 human breast cancer cells after proteasome inhibition

The eukaryotic proteasome is a 26S ATP-dependent proteolytic complex, which possesses chymotrypsin-like, trypsin-like and peptidyl glutamyl peptide hydrolase (PGPH) activities, which enable the proteasome to degrade all short-lived and many long-lived proteins, and consequently regulate a myriad of activities in cells. In this study, we observed that inhibition of the proteasome, and more specifically, inhibition of the chymotrypsin-like activity of the proteasome, in MCF-7 human breast cancer cells resulted in selective down-regulation of the nuclear estrogen receptor-α (ERα). Our data indicated that estrogen had no effect, whereas the ERα antagonist, tamoxifen, reduced the amount of ERα that could be subjected to down-regulation after proteasome inhibition. Furthermore, our data demonstrated that protein synthesis was required for the down-regulation of ERα to occur. Collectively, these data indicate the existence of a proteasome-dependent mechanism that is utilized by MCF-7 cells to maintain a steady-state level of ERα.

Bovine muscle 20S proteasome: I. Simple purification procedure and enzymatic characterization in relation with postmortem conditions

Over the last decade, several sets of evidence support a possible contribution of the 20S proteasome to the meat tenderizing process. This assumption was emphasized by recent investigations demonstrating that the 20S proteasome was active in the absence of activators and exhibited endo- and exoproteolytic activities, a status often strongly debated before. In the present work, we developed a new rapid and simple purification procedure for muscle 20S proteasome and revisited the physicochemical properties of this complex in relation with the postmortem muscle environmental conditions, i.e. temperature, pH, osmolarity, etc. From a crude extract obtained from freshly excised muscle tissue, reasonable amounts of highly pure proteasome were prepared within a maximum of 4 days using only three chromatography steps. This purified proteasome was used to investigate the effect of pH, temperature, ionic strength and sodium dodecyl sulphate (SDS) on the major hydrolytic activities of this complex, i.e. trypsin-like (TL), chymotrypsin-like (CL) and peptidylglutamyl peptide hydrolase (PGPH) activities. Taken together, the data obtained suggest that the 20S proteasome constitutes a high hydrolytic potential in postmortem muscle conditions. To attest this finding, the 20S proteasome was further quantified by ELISA in at death and postmortem muscles including Longissimus, Rectus abdominis, Diaphragma pedialis and Tensor fascia latae bovine muscles. The primary conclusion was that time course changes in proteasome concentrations were not dependent on the kinetics of the pH fall. Secondly, the proteasome concentration in conditioned meat was in good agreement with previously reported proteolytic activity. Furthermore, the decrease in the muscle proteasome concentration can be considered as slow and this is particularly true in type 1 muscles for which the decrease in the amount of this complex did not exceed 7% during the first three days postmortem. This would suggest that the 20S proteasome was relatively stable during meat conditioning, a feature supporting a potential role in the meat tenderizing process.

Age-dependent protein modifications and declining proteasome activity in the human lens

The proteasome is known to be the main enzymatic complex responsible for the intracellular degradation of altered proteins, and the age-related accumulation of modified lens proteins is associated to the formation of cataracts. The aim of this study was to determine whether the human lens proteasome becomes functionally impaired with age. The soluble and insoluble protein fractions of human lenses corresponding to various age-groups were characterized in terms of their levels of glyco-oxidative damage and found to show increasing anti-carboxymethyl-lysine immunoreactivity with age. Concomitantly, decreasing proteasome contents and peptidase activities were observed in the water-soluble fraction. The fact that peptidylglutamyl–peptide hydrolase activity is most severely affected with age suggests that specific changes are undergone by the proteasome itself. In particular, increasing levels of carboxymethylation were observed with age in the proteasome. It was concluded that the lower levels of soluble active enzymatic complex present in elderly lenses and the post-translational modifications affecting the proteasome may at least partly explain the decrease in proteasome activity and the concomitant accumulation of carboxymethylated and ubiquitinated proteins which occur with age.

Proteasomal Activity in Synaptosomes Obtained from the Cerebral Structures of Rats Subjected to Long-Lasting Immobilization Stress

We studied the proteasomal activity in synaptosomes obtained from tissues of the cerebral cortex, cerebellum, and hippocampus, as well as in the cytoplasm of cells of these brain structures, of rats subjected to long-lasting immobilization stress. It was demonstrated that the chymotrypsin-like activity of proteasomes in synaptosomes of the cerebral cortex and hippocampus of stressed animals was significantly higher (380 and 560%, respectively) as compared with that observed in control rats. The chymotrypsin-like and peptidylglutamyl peptide hydrolase activities of proteasomes in the cytoplasm of cortical cells under stress conditions also increased (210 and 180%, respectively). These data show that the activity of a multicatalytic proteolytic complex is sharply increased in synaptic terminals of cells of the cerebral cortex and hippocampus of stressed animals. The above complex plays a crucial role in the utilization of short-lived proteins whose molecules form receptors and ion channels; the amount of such proteins is especially great in synaptic terminals.

Short-term CR decreases cardiac mitochondrial oxidant production but increases carbonyl content.

Lifelong caloric restriction (CR) reduces the rate of mitochondrial oxidant production and the accumulation of oxidized proteins and prevents some of the age-associated decline in 20S proteasome activity. However, few studies have investigated how rapidly the beneficial effects of CR take place. We investigated whether 2 mo of CR in 6-mo-old rats would be of sufficient duration to elicit these beneficial changes. Mitochondrial oxidant production was significantly diminished in the CR rats compared with the ad libitum-fed animals. Short-term CR also caused a significant decrease in mitochondrial superoxide dismutase (SOD) and glutathione peroxidase (GPX) activities, but there were no differences in cytosolic SOD and GPX activities, whereas mitochondrial and cytosolic catalase (CAT) activity was increased with CR. However, protein carbonyl content was significantly elevated in both the mitochondrial and cytosolic fractions from CR rats. Of the three major 20S proteasome activities (chymotrypsin-like, trypsin-like, and peptidylglutamyl-peptide hydrolase), the peptidylglutamyl-peptide hydrolase activity was significantly elevated in the CR animals, possibly because of the fact that there were more oxidized proteins to be degraded. Although fewer oxidants were produced in the CR animals, it is possible that the ability to scavenge oxidants was transiently suppressed because of the reduction in mitochondrial antioxidant enzyme activities, which may explain the observed increases in carbonyl content.

Increased muscle proteasome activities in rats fed a polyunsaturated fatty acid supplemented diet

Changes in the proteasome system, a dominant actor in protein degradation in eukaryotic cells, have been documented in a large number of physiological and pathological conditions. We investigated the influence of monounsaturated or polyunsaturated fatty acids (PUFAs) supplemented diets on the proteasome system, in rat skeletal muscles. Thirty rats were randomly assigned to three groups. The control group received only a standard diet. The monounsaturated fatty acid (MUFA) enriched diet group was fed with 3% sunflower oil in addition to standard food, and the polyunsaturated fatty acid supplemented diet group received 9% Maxepa ® in addition to the standard diet. We analyzed muscle proteasome activities and content. Monounsaturated or PUFAs supplemented diets given for 8 weeks induced a significant increase in proteasome activities. With the polyunsaturated fatty acid enriched diet, the chymotrypsin-like and peptidylglutamylpeptide hydrolase activities increased by 45% in soleus and extensor digitorum longus (EDL), and by 90% in the gastrocnemius medialis (GM) muscle. Trypsin-like activity of the proteasome increased by 250% in soleus, EDL and GM. This increase in proteasome activities was associated with a concomitant enhancement in the muscle content of proteasome. Proteasome activities and level were less stimulated with a monounsaturated fatty acid supplemented diet. This study provides evidence that a monounsaturated or polyunsaturated fatty acid supplemented diet may regulate muscle proteasomes. Unsaturated fatty acids are particularly prone to free radical attack. Thus, we suggest that alterations in muscle proteasome may result from monounsaturated and polyunsaturated fatty acid-induced peroxidation, in order to eliminate damaged proteins.

Peptidase activities of the 20/26S proteasome and a novel protease in human brain.

Many neurodegenerative diseases are characterized by ubiquitin-positive protein aggregates or inclusion bodies. Ubiquitin-conjugated proteins are degraded by the 20/26S proteasome, and reduced proteasome peptidase activities in brain homogenates have been reported in pathologic lesions of Parkinson's and Alzheimer's diseases. However, it is unknown whether crude extracts of human brain contain other proteases having peptidase activities. We found a novel protease of molecular weight of approximately 105 kDa in normal human brain, which exhibited trypsin-like (T-L) and chymotrypsin-like (ChT-L) activities (corresponding to 52% and 21% of the total activities in crude extracts) but not peptidyl glutamyl peptide hydrolase activity. Both T-L and ChT-L activities of this protease were partially inhibited by proteasome inhibitors (MG132, lactacystin) and, in contrast to those of the proteasome, also by sodium dodecyl sulfate. A simple method to obtain a brain fraction specific to the 20/26S proteasome was developed. Our human brain data suggest that T-L and ChT-L activity levels of the proteasome reported previously may include those of the 105 kDa protease, an enzyme of as yet unknown biological significance, and that it is necessary to separate the proteasome from this protease to evaluate the actual status of the ubiquitin-proteasome system in neurodegenerative disorders.

Effect of neurotoxic metal ions on the proteolytic activities of the 20S proteasome from bovine brain.

The effect of oxidative stress induced by neurotoxic metal ions on the properties of the brain 20S proteasome or multicatalytic proteinase complex (MPC) has been studied. Exposure of the 20S proteasome to increasing amounts of Fe(III), Fe(II), Cu(II) or Zn(II) affects its main hydrolytic activities: trypsin-like (T-L), chymotrypsin-like (ChT-L), peptidylglutamyl-peptide hydrolase (PGPH), branched-chain amino acid preferring (BrAAP) and caseinolytic activities, although in different ways. T-L activity showed gradual activation by both iron ions but inhibition by Cu(II) and Zn(II). ChT-L and PGPH activities were inhibited whereas BrAAP activity was widely activated by all the tested metal salts except for zinc ions. Moreover, the exposure to ferrous salt increased the degradation rate of casein. The functional effects appear to be linked to oxidation-induced modifications, as demonstrated by an increase of carbonyl groups following the exposure to metal ions. In addition, modifications induced by ferrous salt on the catalytic subunits were also supported by western blot analyses performed using anti-X, anti-Y and anti-Z antibodies. The results obtained clearly indicate that metal-catalyzed oxidation strongly affects the functions of the brain 20S proteasome, even though the catalytic subunits seem to be differently influenced by oxidative phenomena.

The core structure of TMC-95A is a promising lead for reversible proteasome inhibition.

Correspondingly,great attention has recently been paid to the discovery ofpotentandselectiveproteasomeinhibitorsbystructure-baseddesignornaturalproductscreeningapproaches.Mostofthesynthetic inhibitors consisting of peptide aldehydes, boro-nates, and vinylsulfones, as well as the natural productslactacystinandepoxymicinsinhibitinamoreorlessselectivemanner the proteasome by reaction with the N-terminalthreonineresidue(forarecentreviewseeref.[5]).Anotableexceptionisthehighlyselectiveandcompetitiveproteasomeinhibitor TMC-95A, which was isolated from the fermenta-tionbrothofApiosporamontagneiSacc.TC1093.

Oxidative Modification and Inactivation of the Proteasome during Coronary Occlusion/Reperfusion

Restoration of blood flow to ischemic myocardial tissue results in an increase in the production of oxygen radicals. Highly reactive, free radical species have the potential to damage cellular components. Clearly, maintenance of cellular viability is dependent, in part, on the removal of altered protein. The proteasome is a major intracellular proteolytic system which degrades oxidized and ubiquitinated forms of protein. Utilizing an in vivo rat model, we demonstrate that coronary occlusion/reperfusion resulted in declines in chymotrypsin-like, peptidylglutamyl-peptide hydrolase, and trypsin-like activities of the proteasome as assayed in cytosolic extracts. Analysis of purified 20 S proteasome revealed that declines in peptidase activities were accompanied by oxidative modification of the protein. We provide conclusive evidence that, upon coronary occlusion/reperfusion, the lipid peroxidation product 4-hydroxy-2-nonenal selectively modifies 20 S proteasome alpha-like subunits iota, C3, and an isoform of XAPC7. Occlusion/reperfusion-induced declines in trypsin-like activity were largely preserved upon proteasome purification. In contrast, loss in chymotrypsin-like and peptidylglutamyl-peptide hydrolase activities observed in cytosolic extracts were not evident upon purification. Thus, decreases in proteasome activity are likely due to both direct oxidative modification of the enzyme and inhibition of fluorogenic peptide hydrolysis by endogenous cytosolic inhibitory protein(s) and/or substrate(s). Along with inhibition of the proteasome, increases in cytosolic levels of oxidized and ubiquitinated protein(s) were observed. Taken together, our findings provide insight into potential mechanisms of coronary occlusion/reperfusion-induced proteasome inactivation and cellular consequences of these events.

Increase of oxidatively modified protein is associated with a decrease of proteasome activity and content in aging epidermal cells.

For the process of aging in epidermal cells to be characterized, the status of oxidized and damaged protein accumulation and removal by the proteasome has been investigated. Modified protein content and proteasome activity were assayed in lysates of epidermal cells from donors of different ages. Increased levels of oxidized proteins, glycated proteins, and proteins modified by the lipid peroxidation product 4-hydroxy-2-nonenal were observed in cells from old donors. At the same time, a decline of chymotrypsin-like and peptidylglutamyl-peptide hydrolase activities of the proteasome was found in aging keratinocytes. This age-related decline of the proteasome peptidase activities can be explained, at least in part, by a decreased proteasome content as observed by immunoblotting and enzyme-linked immunosorbent assay. In keratinocyte cultures, a decrease of proteasome activity and content was observed upon serial passaging. In cultures, as well as in skin, an inverse relationship was found between the aging marker 1-galactosidase and the proteasome content. These results suggest that proteasome is downregulated during replicative senescence as well as in aged cells in vivo, possibly resulting in the accumulation of modified proteins.

4-Hydroxy-2-nonenal-mediated Impairment of Intracellular Proteolysis during Oxidative Stress: IDENTIFICATION OF PROTEASOMES AS TARGET MOLECULES

Oxidative stress is associated with important pathophysiological events in a variety of diseases. It has been postulated that free radicals and lipid peroxidation products generated during the process may be responsible for these effects because of their ability to damage cellular components such as membranes, proteins, and DNA. In the present study, we provide evidence that oxidative stress causes a transient impairment of intracellular proteolysis via covalent binding of 4-hydroxy-2-nonenal (HNE), a major end product of lipid peroxidation, to proteasomes. A single intraperitoneal treatment with the renal carcinogen, ferric nitrilotriacetate, caused oxidative stress, as monitored by accumulation of lipid peroxidation products and 8-hydroxy-2'-deoxyguanosine, in the kidney of mice. In addition, transient accumulation of HNE-modified proteins in the kidney was also found by competitive enzyme-linked immunosorbent assay and immunohistochemical analyses. This and the observation that the HNE-modified proteins were significantly ubiquitinated suggested a crucial role of proteasomes in the metabolism of HNE-modified proteins. In vitro incubation of the kidney homogenates with HNE indeed resulted in a transient accumulation of HNE-modified proteins, whereas the proteasome inhibitor significantly suppressed the time-dependent elimination of HNE-modified proteins. We found that, among three proteolytic activities (trypsin, chymotrypsin, and peptidylglutamyl peptide hydrolase activities) of proteasomes, both trypsin and peptidylglutamyl peptide hydrolase activities in the kidney were transiently diminished in accordance with the accumulation of HNE-modified proteins during oxidative stress. The loss of proteasome activities was partially ascribed to the direct attachment of HNE to the protein, based on the detection of HNE-proteasome conjugates by an immunoprecipitation technique. These results suggest that HNE may contribute to the enhanced accumulation of oxidatively modified proteins via an impairment of ubiquitin/proteasome-dependent intracellular proteolysis.