Proteasome activity in tumors of female reproductive system

Proteasomes (multiproteinase protein complexes) are known to play an important role in cancer pathogenesis, however, few information about their activity in human tumor tissues is available so far. We studied chymotrypsin-like activity of proteasomes in tissues of breast cancer (BC) and endometrial cancer (EC). The chymotrypsin-like total proteasome activity and the 20S and 26S proteasome activity in malignant tissues were shown to be significantly higher in malignant tumors than in normal tissues. No increase in proteasome activity was registered with larger tumor size in both BC and EC, whereas proteasome activity was changed with respect to the extent of tumor involvement. In breast cancer tissues, significant reductions in the total and the 26S proteaome activities were observed in tumors with regional lymph node metastases as compared to tumors without metastases. In endometrial cancer tissues, the total proteasome activity and the 20S and 26S proteasome activities were increased as the depth of myometrial invasion. The data obtained indicate that the proteasome acyivity is significantly changed in the process of cancerogenesis and further study is needed to develop new additional prognostic criteria and effective anti-tumor agents in molecular-directed therapy.     

Chymotrypsin-like activity and subunit composition of proteasomes in human cancers

The activity of the proteasome, a polyfunctional enzymatic complex, is known to undergo changes during cancer development. This phenomenon is probably caused by the changes in subunit composition of proteasomes. In this work, we studied the chymotrypsin-like activity of proteasomes; their subunit composition; and their association in breast cancer, head and neck squamous cell carcinoma, endometrial cancer, renal cancer, bladder cancer, stomach cancer, and colorectal cancer. The increase in proteasome activity was revealed in most cancer tissues compared with adjacent tissues, except for the renal cell carcinoma. Changes in proteasome activity in cancer tissues compared with correspondent normal tissues observed in combination with an increased expression of immune subunits and/or proteasome activator PA28β associated with activity of 20S proteasome. In breast cancer, head and neck squamous cell carcinoma, bladder cancer, stomach cancer, and colorectal cancer, we additionally found the higher expression of Rpt6 subunit of the 19S-subunit in 26S proteasome. Correlations between chymotrypsin-like proteasome activity and subunit expressions were found in human cancer tissues. Thus, we suggest that proteasome activation and changes in its subunit composition play an important role in cancer pathogenesis.     

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.     

Expression and activity of proteases in metastasis of ovarian cancer

The total chymotrypsin-like activity of proteasomes, the activity of 20S- and 26S-proteasome pools and calpains, and the expression of metalloproteinase PAPP-A in primary tumors and metastasized tissues were studied in 13 patients with epithelial ovarian cancer. It was shown that initiation of the process of tumor dissemination occurs against the background of active proteolytic processes. A decrease in activity of 26S-proteasomes and total calpain activity and increased expression of metalloproteinase PAPP-A in the primary tumors were found in patients with ascites as compared with patients without ascites. The disease progression after treatment and achieved stabilization were found in patients with decreased activity of intracellular proteases and a high content of PAPP-A in the primary tumors.     

Association between intracellular proteinase activities and the content of locomotor proteins in tissues of primary tumors and metastases of ovarian cancer

The capability for active movement in an extracellular matrix, wherein remodeling of the cytoskeleton by actin-binding proteins plays a significant role, may influence the metastatic potential of tumor cells. We studied the expression of actin-binding proteins and β-catenin in connection with proteasome and calpain functions in tissues of primary tumors and metastases of ovarian cancer. The chymotrypsin-like proteasome activity and calpain activity were shown to be significantly higher in ovarian cancer than in normal tissues. Furthermore, the activity of the proteasome and calpain were significantly higher in the peritoneal metastases in comparison with primary tumors. Correlation analysis showed the presence of a positive correlation between the activity of calpain and chymotrypsin-like proteasome activity (r = 0.82; p = 0.0005) in primary tumor tissue, whereas no such correlation was revealed in metastases. Contents of p45 Ser β-catenin and the actin-severing protein gelsolin were decreased in metastases relative to primary tumors. The level of the cofilin, functionally similar to gelsolin, was significantly higher in metastases compared to primary ovarian tumor tissue. In ovarian cancer, significant reduction in the number of an actin-monomer binder protein thymosin-β4 was observed in primary tumors and metastases as compared to normal tissues, but no significant differences between primary tumors and metastases were observed. In tissues of primary tumors, negative correlations were observed between the chymotrypsin-like activity of proteasomes and the amounts of p45 Ser β-catenin and protein Arp3, a member of the Arp2/3 complex. In metastasis, negative correlation was revealed between the activity of calpain and the content of Arp3, cofilin, and thymosin. The data obtained suggest that the mechanisms of proteolytic regulation of locomotor proteins in primary tumors and metastases in ovarian cancer are different.     

Pattern of MHC class I and immune proteasome expression in Walker 256 tumor during growth and regression in Brattleboro rats with the hereditary defect of arginine-vasopressin synthesis

Dynamics of the expression of MHC class I, immune proteasomes and proteasome regulators 19S, PA28, total proteasome pool and proteasome chymotrypsin-like activity in Walker 256 tumor after implantation into Brattleboro rats with the hereditary defect of arginine-vasopressin synthesis was studied. The tumor growth and regression in Brattleboro rats were accompanied by changes in the proteasome subunit level unlike the tumor growth in WAG rats with normal expression of arginine-vasopressin gene. In the tumor implanted into Brattleboro rats the immune proteasome level was maximal between days 14 and 17, when the tumor underwent regression. Conversely, the expression of proteasome regulators tended to decrease during this period. Immune proteasomes are known to produce antigen epitopes for MHC class I to be presented to CD8+ T lymphocytes. Enhanced expression of immune proteasomes coincided with the recovery of MHC class I expression, suggesting the efficient presentation of tumor antigens in Brattleboro rats.     

Peculiarities of proteasome pool formation in rat spleen and liver during postnatal development

Changes in the specific activity and amounts of 26S and 20S proteasome pools in rat spleen and liver during postnatal development and appearance in them of immune subunits were studied. Two decreases in chymotrypsin-like activity of the proteasome pools were recorded during the first three weeks after birth. The activity minimum fell on the 11th and 19th days, and the first decrease was more prolonged and pronounced than the second. The decrease in the specific activity of the 26S proteasome pools was associated with a reduction of their quantity. The 20S proteasome pools displayed no such decreases. Noticeable quantities of immune subunits LMP7 and LMP2 were revealed by Western blotting in the spleen on the 7th day and on the 19th day in the liver, concurrently with the beginning of the decrease in the proteasome activity. It was concluded that during the first three weeks of postnatal development the proteasome pools in rat spleen and liver were replaced twice, and in the spleen (a lymphoid organ) a qualitatively new pool containing immune subunits appeared nearly two weeks earlier than in the liver (a non-lymphoid organ). The appearance of immune proteasomes in different organs and tissues during some weeks after birth seems to explain the immune system inefficiency during embryogenesis and early postnatal development.     

Exclusion of immune proteasomes from mouse ascitic carcinoma Krebs-II cells

Pools of 26S and 20S proteasomes were studied in the spleen, liver, lung, and ascitic carcinoma Krebs-II of mouse. Western blotting demonstrated that the pool of 26S proteasomes in ascitic carcinoma Krebs-II was twice that in control lung cells and did not significantly differ by total 26S proteasome quantities from the spleen and liver. At the same time, the level of immune subunit LMP7 was 12 times lower in it compared to lung proteasomes and 4–5 times lower compared to spleen and liver proteasomes. Immune subunit LMP2 was undetectable by this technique in the ascitic carcinoma in contrast to the lung, spleen, and liver. All immune subunits in the studied organs and ascitic carcinoma Krebs-II are components of 26S but not 20S proteasomes.     

Different proteasome subtypes in a single tissue exhibit different enzymatic properties

It is concluded from many experiments that mammalian tissues and cells must contain a heterogeneous population of 20 S proteasome complexes. We describe the purification and separation by chromatographic procedures of constitutive 20 S proteasomes, 20 S immuno-proteasomes and intermediate-type 20 S proteasomes from a given tissue. Our data demonstrate that each of these three groups comprises more than one subtype and that the relative ratios of the subtypes differ between different rat tissues. Thus, six subtypes could be identified in rat muscle tissue. Subtypes I and II are constitutive proteasomes, while subtypes V and VI comprise immuno-proteasomes. Subtypes III and IV belong to a group of intermediate-type proteasomes. The subtypes differ with regard to their enzymatic characteristics. Subtypes I-III exhibit high chymotrypsin-like activity and high peptidylglutamylpeptide hydrolysing activity, while these activities are depressed in subtypes IV-VI. In contrast, trypsin-like activity of subtypes IV-VI is enhanced in comparison to subtypes I-III. Importantly, the subtypes also differ in their preferential cleavage site usage when tested by digestion of a synthetic 25mer polypeptide substrate. Therefore, the characteristics of proteasomes purified from tissues or cells represent the average of the different subtype activities which in turn may have different functions in vivo.     

Changes in proteasome pool in human papillary thyroid carcinoma development

Searching the antitumor drug targets among proteasomes, “ubiquitous” enzyme systems, may provide a new impulse to the antitumor drug discovery. In this study, changes in the proteasome pool in the development of human papillary thyroid carcinoma were determined. Proteasome activities were evaluated by hydrolysis of commercial fluorogenic peptides. Changes in the expression of the total proteasome pool, proteasome 19S activator and proteolytic constitutive subunits X(β5), Y(β1) and immune subunits LMP7 (β5i) and LMP2 (β1i) were investigated by Western blotting. The distribution of the proteasome subunits in thyroid gland cells was detected by immunohistochemistry. It was shown that the chymotrypsin- and caspase-like activities as well as the expression of the total proteasome pool, proteasome 19S activator and immune subunits increased gradually in the tumors at the T₂N₀M₀ and T₃N₀M₀ stages in comparison with the control tissues. Among the structures studied, the expression of the 19S activator and immune proteasomes, which contain the LMP2 (β1i) subunit, was enhanced to the largest degree in tumor cells. The data obtained may be implicated in a new therapeutic strategy. Taking into consideration the antitumor function of the immune proteasomes, we advance the 19S activator as the target for the development of a novel antitumor therapy.     

Proteasome active sites allosterically regulate each other, suggesting a cyclical bite-chew mechanism for protein breakdown.

In eukaryotes, the 20S proteasome contains two chymotrypsin-like, two trypsin-like, and two active sites shown here to have caspase-like specificity. We report that certain sites allosterically regulate each other's activities. Substrates of a chymotrypsin-like site stimulate dramatically the caspase-like activity and also activate the other chymotrypsin-like site. Moreover, substrates of the caspase-like sites inhibit allosterically the chymotrypsin-like activity (the rate-limiting one in protein breakdown) and thus can reduce the degradation of proteins by 26S proteasomes. These allosteric effects suggest an ordered, cyclical mechanism for protein degradation. We propose that the chymotrypsin-like site initially cleaves ("bites") the polypeptide, thereby stimulating the caspase-like sites. Their activation accelerates further cleavage ("chewing") of the fragments, while the chymotrypsin-like activity is temporarily inhibited. When further caspase-like cleavages are impossible, the chymotryptic site is reactivated and the cycle repeated.     

Hyperphosphorylation of rat liver proteasome subunits: the effects of ethanol and okadaic acid are compared

In experimental alcoholic liver disease, protein degradation by the ATP-ubiquitin-proteasome pathway is inhibited. Failure of the proteasome to eliminate cytoplasmic proteins leads to the accumulation of oxidized and otherwise modified proteins. One possible explanation for the inhibition of the proteasome is hyperphosphorylation of proteasome subunits. To examine this possibility, the 26S proteasomes from the liver of rats fed ethanol and a pair-fed control were studied by isolating the proteasomes in a purified fraction. The effect of ethanol on the phosphorylation of proteasomal subunits was compared with the hyperphosphorylation of the proteasomes caused by okadaic acid given to rats in vivo. Ethanol ingestion caused an inhibition of the chymotrypsin-like activity of the purified proteasome. The 2D electrophoresis and Western blot analysis of the purified 20S and 26S proteasomes from the ethanol-fed rats indicated that hyperphosphorylation of proteasomal subunits had occured. The proteasomal alpha type subunits C9/alpha3 and C8/alpha7 were hyperphosphorylated compared to the controls. Chymotrypsin-like activity was also inhibited by okadaic acid treatment similar to ethanol feeding. The 26S proteasome fraction examined by isoelectric focusing gel revealed many hyperphosphorylated bands in the proteasomes from the okadaic acid treated and the ethanol fed rat livers compared with the controls. In conclusion hyperphosphorylation of the proteasome subunits occurs in the ethanol treated proteasomal subunits which could be one mechanism of the inhibition of the 26S proteasome caused by ethanol feeding.     

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 beta subunits. CRF rat 26S proteasomes had decreased levels of beta1, beta3, alpha4, and alpha7 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.     

Binding of hydrophobic peptides to several non-catalytic sites promotes peptide hydrolysis by all active sites of 20 S proteasomes. Evidence for peptide-induced channel opening in the alpha-rings

The eukaryotic 20 S proteasome contains the following 6 active sites: 2 chymotrypsin-like, 2 trypsin-like, and 2 caspase-like. We previously showed that hydrophobic peptide substrates of the chymotrypsin-like sites allosterically stimulate peptide hydrolysis by the caspase-like sites and their own cleavage. More thorough analysis revealed that these peptides also stimulate peptide hydrolysis by the trypsin-like site. This general activation by hydrophobic peptides occurred even if the chymotrypsin-like sites were occupied by a covalent inhibitor and was highly cooperative, with an average Hill coefficient of 7. Therefore, this stimulation of peptide hydrolysis at all active sites occurs upon binding of hydrophobic peptides to several non-catalytic sites. The stimulation by hydrophobic peptides was not observed in the yeast Delta N alpha 3 mutant 20 S proteasomes, in 20 S-PA26 complexes, or SDS-activated proteasomes and was significantly lower in 26 S proteasomes, all of which appear to have the gated channel in the alpha-rings in an open conformation and hydrolyze peptides at much faster rates than 20 S proteasomes. Also the hydrophobic peptides altered K(m), V(max) of active sites in a similar fashion as PA26 and the Delta N alpha 3 mutation. The activation by hydrophobic peptides was decreased in K(+)-containing buffer, which favors the closed state of the channels. Therefore, hydrophobic peptides stimulate peptide hydrolysis most likely by promoting the opening of the channels in the alpha-rings. During protein breakdown, this peptide-induced channel opening may function to facilitate the release of products from the proteasome.     

Diagnostics of thyroid cancer: Limitations of the existing methods and perspectives for future developments

A novel approach to the development of a precise method of intraoperative diagnostics of thyroid cancer has been proposed on the basis of fundamental study of proteasomes in malignant tumors of mammals and human. The method is based on estimation of proteasome activity in small fragments of the tumor and adjacent tissues.     

Proteasome structures affected by ionizing radiation

Exposure of cells to ionizing radiation slows the rate of degradation of substrates through the proteasome. Because the 26S proteasome degrades most short-lived cellular proteins, changes in its activity might significantly, and selectively, alter the life span of many signaling proteins and play a role in promoting the biological consequences of radiation exposure, such as cell cycle arrest, DNA repair, and apoptosis. Experiments were therefore undertaken to identify the radiation target that is associated with the proteasome. Regardless of whether they were irradiated before or after extraction and purification from human prostate cancer PC3 cells, 26S proteasomes remained intact but showed a rapid 30% to 50% dose-independent decrease in their three major enzymatic activities following exposure to 1 to 20 Gy. There was no effect on 20S proteasomes, suggesting that the radiation-sensitive target is located in the 19S cap of the 26S proteasome, rather than in the enzymatically active core. Because the base of the 19S cap contains an ATPase ring that mediates substrate unfolding, pore opening, and translocation of substrates into the catalytic chamber, we examined whether the ATPase activity of purified 26S proteasomes was affected. In fact, in vitro irradiation of proteasomes enhanced their ATPase activity. Furthermore, pretreatment with low concentrations of the free radical scavenger tempol was able to prevent both the radiation-induced decrease in proteolytic activity and the increase in ATP utilization, indicating that free radicals are mediators of these radiation-induced phenomena. Finally, we have shown that cell irradiation results in the accumulation of proteasome substrates: polyubiquitinated proteins and ornithine decarboxylase, indicating that the observed decrease in proteasome function is physiologically relevant.     

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.