Proteasome Peptidase Activities Research Articles

Proteasomes in Human Bronchoalveolar Lavage Fluid After Burn and Inhalation Injury

The purpose of this study was to determine whether 26S proteasome is detectable in human bronchoalveolar lavage fluid (BALF) and whether burn and inhalation injury is accompanied by changes in BALF proteasome content or activity. BALF was obtained on hospital admission from 28 patients with burn and inhalation injury (controls: 10 healthy volunteers). Proteasome concentrations were quantified by enzyme-linked immunosorbent assay, and their native molecular mass was assessed by gel filtration. Proteasome peptidase activity was measured using a chymotryptic-like peptide substrate in combination with epoxomicin (specific proteasome inhibitor). BALF protein was increased in patients (P<.001) and correlated positively with the degree of inhalation injury. The 20S/26S proteasomes were detectable in all BALF by enzyme-linked immunosorbent assay. Gel filtration confirmed the presence of intact 20S and 26S proteasome that was stable without soluble ATP/Mg. In all BALF chymotryptic-like activity was detectable and could be inhibited with epoxomicin by 60 to 70% (P<.01). Absolute amounts of 20S/26S proteasomes and proteasome activity were increased in patients (P<.001 for all). The relative BALF composition after injury was characterized by increased concentrations of 20S proteasome/mg protein (P=.0034 vs volunteers), decreased concentrations of 26S proteasome/mg protein (P=.041 vs volunteers), and reduced specific proteasome activity (P=.044 vs volunteers). The 26S proteasome per milligram and specific proteasome activity were even further reduced in patients who developed ventilator-associated pneumonia (P=.045 and P=.03 vs patients without ventilator-associated pneumonia). This study supports the novel concept that extracellular proteasomes could play a pathophysiological role in the injured lung and suggests that insufficient proteasome function may increase susceptibility for pulmonary complications.

Characterization of a REG/PA28 Proteasome Activator Homolog in Dictyostelium discoideum Indicates that the Ubiquitin- and ATP-Independent REGγ Proteasome Is an Ancient Nuclear Protease

The nuclear proteasome activator REGgamma/PA28gamma is an ATP- and ubiquitin-independent activator of the 20S proteasome and has been proposed to degrade and thereby regulate both a key human oncogene, encoding the coactivator SRC-3/AIB1, and the cyclin-dependent kinase inhibitor p21 (Waf/Cip1). We report the identification and characterization of a PA28/REG homolog in Dictyostelium. Association of a recombinant Dictyostelium REG with the purified Dictyostelium 20S proteasome led to the preferential stimulation of the trypsin-like proteasome peptidase activity. Immunolocalization studies demonstrated that the proteasome activator is localized to the nucleus and is present in growing as well as starving Dictyostelium cells. Our results indicate that the Dictyostelium PA28/REG activator can stimulate both the trypsin-like and chymotrypsin-like activities of the 20S proteasome and supports the idea that the REGgamma-20S proteasome represents an early unique nuclear degradation pathway for eukaryotic cells.

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.

Polyubiquitin substrates allosterically activate their own degradation by the 26S proteasome

The 26S proteasome degrades polyubiquitylated (polyUb) proteins by an ATP-dependent mechanism. Here we show that binding of model polyUb substrates to the 19S regulator of mammalian and yeast 26S proteasomes enhances the peptidase activities of the 20S proteasome about two-fold in a process requiring ATP hydrolysis. Monoubiquitylated proteins or tetraubiquitin alone exert no effect. However, 26S proteasomes from the yeast alpha3DeltaN open-gate mutant and the rpt2YA and rpt5YA mutants with impaired gating can still be activated (approximately 1.3-fold to 1.8-fold) by polyUb-protein binding. Thus, binding of polyUb substrates to the 19S regulator stabilizes gate opening of the 20S proteasome and induces conformational changes of the 20S proteasome that facilitate channeling of substrates and their access to active sites. In consequence, polyUb substrates will allosterically stimulate their own degradation.

PROTEASOME PEPTIDASE ACTIVITIES PARALLEL HISTOMORPHOLOGICAL AND FUNCTIONAL CONSEQUENCES OF ISCHEMIA-REPERFUSION INJURY IN THE LUNG

Several lines of evidence suggest that the proteasome contributes to ischemia-reperfusion injury (I-RI) of organs. Although I-RI contributes to multiple disease processes in the lung, the regulation of proteasome activities during pulmonary I-RI is unknown. Thus, the authors performed a pilot study to define time-related changes of lung proteasome peptidase activities and to evaluate if possible alterations correspond to morphological and functional consequences of I-RI using a rat model. Animals underwent 120 minutes of unilateral lung ischemia. Ischemic and contralateral lungs were harvested at multiple time points for up to 168 hours of reperfusion (I-R30min-168h). Chymotryptic-like (CT-L) and tryptic-like (T-L) proteasome peptidase activities were measured in lung extracts. An early I-R–associated inactivation of proteasome activities paralleled impairment of oxygenation, edema formation, and degree of histopathology, and resolved with restoration of function within 24 to 72 hours. Although functional and histomorphological baseline conditions were still not fully achieved at I-R168h, proteasome activities increased continuously 1.4-fold (CT-L) and 5.7-fold (T-L) until I-R168h. Apparent KM values for the CT-L/T-L substrates were not influenced by I-R. This pilot study establishes an initial link between proteasome activities and physiological relevant consequences of lung I-RI, and further points towards a possible role of the proteasome during the postischemic tissue repair process.

Trypanocidal activity of the proteasome inhibitor and anti-cancer drug bortezomib

The proteasome inhibitor and anti-cancer drug bortezomib was tested for in vitro activity against bloodstream forms of Trypanosoma brucei. The concentrations of bortezomib required to reduce the growth rate by 50% and to kill all trypanosomes were 3.3 nM and 10 nM, respectively. In addition, bortezomib was 10 times more toxic to trypanosomes than to human HL-60 cells. Moreover, exposure of trypanosomes to 10 nM bortezomib for 16 h was enough to kill 90% of the parasites following incubation in fresh medium. However, proteasomal peptidase activities of trypanosomes exposed to bortezomib were only inhibited by 10% and 30% indicating that the proteasome is not the main target of the drug. The results suggest that bortezomib may be useful as drug for the treatment of human African trypanosomiasis.

Effect of copper intoxication on rat liver proteasome activity: Relationship with oxidative stress

Copper toxicity is associated with formation of reactive oxygen species, which are capable to oxidize proteins. The selective removal of the latter by the 20S proteasome is considered an essential part of the cell antioxidant defense system. The aim of the present study was to investigate whether peptidase activities of rat liver proteasomes were affected by chronic (40 mg CuSO(4)/rat/daily with the drinking water for 2 weeks) and acute (20 mg/kg CuSO(4), s.c.) copper treatment. To evaluate the role of proteasome, its inhibitor MG132 was also used. The degree of copper-induced oxidative stress (OS), established by measuring lipid peroxidation, protein oxidation, and cellular glutathione level, as well as activities of antioxidant enzymes--catalase, superoxide dismutase, and gultathionine peroxidase, depended on the mode of copper administration. Chronic copper administration (mild oxidative stress) did not affect proteasome activities, whereas acute copper treatment (severe oxidative stress) caused a decline in chymotryptic- and tryptic-like activities. The treatment of copper-loaded animals with MG132 did not change copper-induced alterations in the tested indices, except an additional increase in protein oxidation and inhibition of glutathionine peroxidase activity. The results suggested that the in vivo copper-induced oxidative stress was associated with changes in the catalytic activity of proteasome.

High affinity Zn 2+ inhibitory site(s) for the trypsin-like peptidase of the 20S proteasome

The effect of Zn 2+ on three major peptidase activities of the 20S proteasome purified from Xenopus oocytes was kinetically investigated. An extremely low concentration of Zn 2+ (μM range) strongly inhibited the trypsin-like activity of the 20S proteasome which was fully recoverable by the addition of EDTA. The concentration of Zn 2+ for half-maximum inhibition ( K 0.5) was 0.60 μM which was at least 10 times lower than that of any other divalent cation tested and essentially the same as for proteasomes purified from various other organisms indicating that the inhibition is highly Zn 2+-specific, reversible, and common to the proteasome regardless of its source. Zn 2+ at concentrations below 100 μM instantaneously activated the chymotrypsin-like and PGPH activities, and the Zn 2+ concentration for half-maximum activation was found to be 42–48 μM. These activities were time-dependently inactivated by submillimolar concentrations of Zn 2+. The inactivation rates were dependent on the concentration of Zn 2+ and reached a maximum of 1.60–2.40 min −1 for the three peptidase activities under the conditions used. The Zn 2+ concentration for half-maximum inactivation was found to be 0.70–1.23 mM. This time-dependent inactivation was not reversed by the addition of EDTA or DTT and might not be accompanied by the dissociation of subunits of the 20S proteasome indicating that all activities are inactivated by an identical phenomenon. These results reveal the three types of effects of Zn 2+ on the 20S proteasome.

Enzymatic activity of circulating proteasomes correlates with clinical behavior in patients with chronic lymphocytic leukemia

The ubiquitin-proteasome pathway has been implicated in the pathogenesis of many hematologic malignancies. The authors measured proteasome peptidase activity levels in plasma samples from 225 patients with chronic lymphocytic leukemia (CLL) and correlated the results with clinical behavior. By using fluorogenic kinetic assays, the enzymatic activity levels of 3 proteasomes were measured: chymotrypsin-like (Ch-L), trypsin-like (Tr-L), and caspase-like (Cas-L). All activity levels were significantly higher in patients who had CLL compared with the levels in a control group of healthy volunteers (P<.001). Rai stage was correlated with Ch-L activity (P<.001) but not with Cas-L or Tr-L activity. Levels of beta2 microglobulin (B2M) were correlated with Ch-L activity (correlation coefficient [R]=0.4; P<.001) and with Cas-L activity (R=0.25; P=.001) but not with Tr-L activity. Cas-L activity as a continuous variable was a strong predictor of survival. Ch-L and Cas-L activity levels as categorical variables both were strong predictors of survival; Cas-L activity was independent of B2M level but not of immunoglobulin variable heavy chain gene (IgVH) mutation status. However, the combination of elevated B2M levels (>3.2 mg/L) and Cas-L activity (>1.32 pmoL/second/mL plasma) was associated with significantly shorter survival independent of IgVH mutation status. The current results indicated that measuring plasma proteasome activity has prognostic value in CLL that, when combined with B2M, can be independent of IgVH mutation status.

The switch from the standard proteasome to the immuno proteasome in the mononuclear cells of children with vertically acquired HCV infection

Background The mechanisms responsible for the HCV escape to the immune system remain largely unknown. A strict interaction between NS3, a non-structural protein of HCV, and the LMP-7, a subunit of the immunoproteasome, has been described suggesting that this may result in an impaired antigen presentation. Proteasomes are intracellular multicatalytic complexes whose primary function is the degradation of abnormal or foreign proteins. In particular, they regulate the presentation of viral antigens onto MHC I molecules. The 26S proteasome has a 20S catalytic core and two 19S regulatory complexes. Three subunits (β1, β2 and β5) are responsible for the main proteasomal peptidase activities (PGPH, trypsin-like, and chymotrypsin-like activity, respectively). With certain cytokines, such as interferon-γ (IFN-γ), the three β subunits of the standard proteasome (PS) are replaced by inducible subunits (LMP-2, MECL-1 and LMP-7) to form the immunoproteasome (IP), which improves the antigen processing and presentation.

Cardiac proteasome dysfunction during cold ischemic storage and reperfusion in a murine heart transplantation model

Recent observations suggest that the ubiquitin–proteasome system (UPS) contributes to the pathophysiology of myocardial ischemia–reperfusion injury. Since its regulation during cold ischemia–reperfusion is unknown, we evaluated the cardiac UPS in a model of heart transplantation in mice. Cardiac ubiquitylation rates and ubiquitin–protein conjugates increased after 3 h of cold ischemia (CI) and normalized post-transplant. 20S proteasome content and proteasome peptidase activities were unchanged after CI. 4 h/24 h post-transplant 20S proteasome concentrations decreased and chymotryptic-like but not tryptic-like proteasome peptidase activity was inactivated. Epoxomicin sensitivity of the proteasome increased 5.7-fold during CI and normalized 4 h/24 h post-transplant. This was accompanied by the disappearance of a 13.5 kDa-ubiquitin-conjugate during CI that could be attenuated by addition of epoxomicin to the preservation fluid. We conclude that substrate specificity of the proteasome changes during cold ischemia and that proteasome inhibition preserves the physiological ubiquitin–protein conjugate pool during organ preservation. Reduced proteasome activity during reperfusion is caused by a decrease in proteasome content and enzyme inhibition.

Activity of Circulating Proteasomes Correlates with Clinical Behavior in Patients with Chronic Myeloid Leukemia.

The ubiquitin-proteasome pathway is implicated in the pathogenesis of many malignancies and is an important regulator of cell proliferation, apoptosis, DNA repair, and the stress response. Free circulating proteasomes have been reported in the plasma and sera of cancer patients. We measured proteasome peptidase activity in the plasma of patients with chronic myeloid leukemia (CML) and correlated these findings with clinical behaviour. Plasma samples from apparently healthy volunteers (n = 92) and patients in the chronic (n = 104) or accelerated/blast phase (n = 56) of CML were analysed with fluorogenic kinetic assays. Using peptide-AMC (7-amino-4-methylcoumaran) substrates, we measured the three reported proteasome enzymatic activities: chymotrypsin-like (Ch-L), trypsin-like (Tr-L), and caspase-like (Cas-L). All proteasome activities were significantly higher in CML patients than in healthy volunteers. Extensive characterization of proteasome activities revealed correlations between levels or ratios of specific peptidase activities and disease phase, progression, aggressiveness, and survival. High Ch-L activity correlated with shorter survival in both CP (P=0.037) and Acc/Bl phase (P=0.047). Patients in CP and a ratio of Cas-L: Tr-L activity >1.48 had significantly shorter survival (P=0.03). Cas-L: Tr-L activity ratio was predictor of survival in Acc/Bl patients as a continuous variable. These findings suggest that proteasome activity in the plasma of CML patients reflects disease activity and can be used as a biomarker for predicting clinical behavior. Studies exploring the role of proteasome inhibitors in CML are therefore warranted.

Mammalian Proteasome Subpopulations with Distinct Molecular Compositions and Proteolytic Activities

The proteasome-dependent protein degradation participates in multiple essential cellular processes. Modulation of proteasomal activities may alter cardiac function and disease phenotypes. However, cardiovascular studies reported thus far have yielded conflicting results. We hypothesized that a contributing factor to the contradicting literature may be caused by existing proteasome heterogeneity in the myocardium. In this investigation, we provide the very first direct demonstration of distinct proteasome subpopulations in murine hearts. The cardiac proteasome subpopulations differ in their molecular compositions and proteolytic activities. Furthermore they were distinguished from proteasome subpopulations identified in murine livers. The study was facilitated by the development of novel protocols for in-solution isoelectric focusing of multiprotein complexes in a laminar flow that support an average resolution of 0.04 pH units. Utilizing these protocols, the majority of cardiac proteasome complexes displayed an isoelectric point of 5.26 with additional subpopulations focusing in the range from pH 5.10 to 5.33. In contrast, the majority of hepatic 20 S proteasomes had a pI of 5.05 and focused from pH 5.01 to 5.29. Importantly proteasome subpopulations degraded specific model peptides with different turnover rates. Among cardiac subpopulations, proteasomes with an approximate pI of 5.21 showed 40% higher trypsin-like activity than those with pI 5.28. Distinct proteasome assembly may be a contributing factor to variations in proteolytic activities because proteasomes with pI 5.21 contained 58% less of the inducible subunit beta 2i compared with those with pI 5.28. In addition, dephosphorylation of 20 S proteasomes demonstrated that besides molecular composition posttranslational modifications largely contribute to their pI values. These data suggest the possibility of mixed 20 S proteasome assembly, a departure from the currently hypothesized two subpopulations: constitutive and immuno forms. The identification of multiple distinct proteasome subpopulations in heart provides key mechanistic insights for achieving selective and targeted regulation of this essential protein degradation machinery. Thus, proteasome subpopulations may serve as novel therapeutic targets in the myocardium.

Protein Aggregation and Proteasome Dysfunction After Brain Ischemia

Protein unfolding and aggregation are dominant early pathogenic events in neurons after brain ischemia. This study used a transient cerebral ischemia model to investigate whether overproduction of unfolded proteins after brain ischemia is a consequence of proteasome dysfunction. Proteasome peptidase activity and proteasome subcellular redistribution and assembly were studied by peptidase activity assay, Western blot analysis, and size-exclusion chromatography. Proteasome peptidase activity, as determined with the peptide substrate succinyl-LLVY-7-amino-4-methylcoumarin, was moderately decreased, and the 26S proteasome was disassembled during the early period of reperfusion after transient brain ischemia. Furthermore, the proteasome subunits, particularly the 19S components, were deposited into the protein aggregate-containing fraction after an episode of transient cerebral ischemia. These results clearly demonstrate that after an episode of brain ischemia, proteasomes are disassembled and aggregated and thus fail to function normally. Deposition of proteasomes into protein aggregates may also indicate that proteasomes attempt to degrade ubiquitin-conjugated proteins (ubiproteins) overproduced after brain ischemia. However, ubiproteins are too numerous to be degraded and trap some of the proteasomes into their aggregates after brain ischemia.

Role of increased expression of the proteasome in the protective effects of sulforaphane against hydrogen peroxide-mediated cytotoxicity in murine neuroblastoma cells

The 26S proteasome is responsible for degradation of abnormal proteins and may play a role in cell survival upon oxidative stress. The indirect antioxidant sulforaphane (SFN) protects animal tissues from chemical toxicants by increasing the expression of several families of Nrf2-regulated genes. The role of induction of the 26S proteasome in cytoprotection by SFN was investigated in murine neuroblastoma Neuro2A cells. SFN enhanced the expression of the catalytic subunits of the proteasome, as well as proteasomal peptidase activities in these cells. Such treatment with SFN protected cells from hydrogen peroxide-mediated cytotoxicity in a manner dependent on proteasomal function. Inhibition of proteasome activities using pharmacological interventions significantly attenuated the protective effects of SFN against hydrogen peroxide cytotoxicity, as well as protein oxidation. Moreover, overexpression of the catalytic subunit PSMB5 enhanced proteasome function and led to elevated resistance against hydrogen peroxide toxicity and extent of protein oxidation compared to blank-plasmid-transfected cells. Pretreatment of PSMB5-overexpressing cells with SFN did not further enhance this resistance. Collectively, these results suggest that the cytoprotective effects of SFN against oxidative stress are in part due to up-regulation of the proteasome system. Therefore, inducers of proteasome expression may ameliorate the accumulation of damaged proteins associated with neurodegeneration and other diseases in whose etiologies protein oxidation plays a role.

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.

Tissue specific increase of the catalytic subunits of the 26S proteasome by indirect antioxidant dithiolethione in mice: Enhanced activity for degradation of abnormal protein

Decreases in the 26S proteasome are related to the toxicities of abnormal protein aggregates and may contribute to pathogenesis of degenerative diseases. Therefore, maintenance of proteasome function can be a novel strategy to protect cells against abnormal protein-mediated toxicity. In the present study, we have demonstrated the tissue specific increase of the catalytic subunits of the proteasome in mice following oral administration of 3 H-1,2-dithiole-3-thione (D3T, 0.5 mmol/kg), which functions as a cancer preventive agent in animal and human studies. Expression of the 20S catalytic core subunits PSMB5, PSMB6, and PSMB7 were increased in liver, lung, small intestine, and colon of mice at 24 h after D3T treatment. Elevated expression of proteasome catalytic subunits led to increases in proteasomal peptidase activities in these tissues. Oral administration of D3T also exerted a pharmacodynamic action in some brain regions of these mice and proteasomal peptidase activities were significantly elevated in the cerebral cortex–hippocampus. Moreover, tissue extracts from D3T-treated mice and cell lysates obtained from D3T-incubated murine neuroblastoma cells exhibited the enhanced capacity to degrade mutant human SOD1G93A protein. These results indicate that the catalytic subunits of the 26S proteasome are inducible in multiple tissues of mouse including brain by exogenous chemical treatment. Increased proteasome expression by inducers may have a role in protection/attenuation of protein aggregate-mediated disorders.

Proteasome and Photoaging

Cellular aging is characterized by the accumulation of oxidatively modified proteins that result, at least in part, from impaired degradation of abnormal proteins. The proteasome is the major intracellular proteolytic system implicated in the removal of abnormal and oxidized proteins. In human epidermal cells, previous studies have evidenced that proteasome function is decreased during aging as well as upon UV irradiation, which is the main component of photoaging. The age-related decline of proteasome activity has been reported to be due to either or both decreased proteasome subunits expression and content, inactivation upon alteration of proteasome subunits, and accumulation of endogenous inhibitors, such as highly oxidized and cross-linked proteins. To gain further insight in the mechanisms that might be implicated in the decreased activity of the proteasome upon photoaging, purified 20S human proteasome has been exposed to UVA- and UVB-irradiation. The effect of such an irradiation on proteasome peptidase activities has been monitored and shown to promote a stimulation or an inhibition of the peptidase activities depending on whether the proteasome is under its latent or a nonphysiological active form. Analysis of the patterns of proteasome subunits by 2D gel electrophoresis has revealed modification for several subunits for UV-irradiated proteasome only in its irreversibly activated form, compared with nonirradiated and irradiated latent forms, indicating that the 20S proteasome is rather resistant to UV irradiation.

Proteasomal chymotrypsin‐like peptidase activity is required for essential functions of human monocyte‐derived dendritic cells

The ubiquitin-proteasome pathway is the principal system for extralysosomal protein degradation in eukaryotic cells, and is essential for the regulation and maintenance of basic cellular processes, including differentiation, proliferation, cell cycling, gene transcription and apoptosis. The 26S proteasome, a large multicatalytic protease complex, constitutes the system's proteolytic core machinery that exhibits different proteolytic activities residing in defined proteasomal subunits. We have identified proteasome inhibitors - bortezomib, epoxomicin and lactacystin - which selectively inhibit the proteasomal beta5 subunit-located chymotrypsin-like peptidase activity in human monocyte-derived dendritic cells (DCs). Inhibition of proteasomal chymotrypsin-like peptidase activity in immature and mature DCs impairs the cell-surface expression of CD40, CD86, CD80, human leucocyte antigen (HLA)-DR, CD206 and CD209, induces apoptosis, and impairs maturation of DCs, as demonstrated by decreased cell-surface expression of CD83 and lack of nuclear translocation of RelA and RelB. Inhibition of chymotrypsin-like peptidase activity abrogates macropinocytosis and receptor-mediated endocytosis of macromolecular antigens in immature DCs, and inhibits the synthesis of interleukin (IL)-12p70 and IL-12p40 in mature DCs. As a functional consequence, DCs fail to stimulate allogeneic CD4(+) and CD8(+) T cells and autologous CD4(+) T cells sufficiently in response to inhibition of chymotrypsin-like peptidase activity. Thus, proteasomal chymotrypsin-like peptidase activity is required for essential functions of human DCs, and inhibition of proteasomal chymotrypsin-like peptidase activity by selective inhibitors, or by targeting beta5 subunit expression, may provide a novel therapeutic strategy for suppression of deregulated and unwanted immune responses.

Distribution and interrelationship of ubiquitin proteasome pathway component activities and ubiquitin pools in various porcine tissues

The ubiquitin-proteasome pathway fulfills major biological functions, but its physiologic tissue distribution and the interrelationship between pathway component activities and ubiquitin pools are unknown. Therefore, we analyzed free and conjugated ubiquitin, ubiquitin-protein ligation rates (UbPL) and chymotryptic- and tryptic-like proteasome peptidase activities in porcine skeletal muscle, heart, lung, liver, spleen and kidney (n=5 each). There were considerable differences between tissues (p<0.05 for all parameters). Lung and spleen showed high levels of free and conjugated ubiquitin and high UbPL. Proteasome activities were highest in kidney and heart. There were linear relationships between tryptic-like and chymotryptic-like proteasome peptidase activities (r(2) = 0.624, p<0.001) and between free and conjugated ubiquitin tissue levels (r(2) = 0.623, p<0.001). Tissue levels of free and conjugated ubiquitin correlated linear with UbPL (p<0.005), but they were not correlated with proteasome peptidase activities. The results suggest that tissue ubiquitin pools are tightly regulated and indicate a constant proportion of conjugated ubiquitin. They further support the hypothesis that ubiquitin-protein ligase systems, and probably deubiquitylating enzymes, are key regulators of ubiquitin homeostasis. The detected differences are suggestive of tissue-specific roles of ubiquitin-proteasome pathway components. Besides the known importance of the ubiquitin proteasome pathway in heart, kidney and the immune system, the results suggest the lung as another organ in which ubiquitin proteasome pathway components may also significantly contribute to disease processes.