Proteasomes in Patient Rectal Cancer and Different Intestine Locations: Where Does Proteasome Pool Change?

A special problem in the surgery of rectal cancer is connected with a need for appropriate removal of intestine parts, along with the tumor, including the fragment close to the sphincter. To determine the length of fragments to remove, it is necessary to reveal areas without changes in molecule functioning, specific for tumor. The purpose of the present study was to investigate functioning the proteasomes, the main actors in protein hydrolysis, in patient rectal adenocarcinoma and different intestine locations. Chymotrypsin-like and caspase-like activities, open to complex influence of different factors, were analyzed in 43-54 samples by Suc-LLVY-AMC- and Z-LLE-AMC-hydrolysis correspondingly. Both activities may be arranged by the decrease in the location row: cancer→adjacent tissue→proximal (8-20 cm from tumor) and distal (2 and 4 cm from tumor) sides. These activities did not differ noticeably in proximal and distal locations. Similar patterns were detected for the activities and expression of immune subunits LMP2 and LMP7 and expression of 19S and PA28αß activators. The largest changes in tumor were related to proteasome subtype containing LMP2 and PA28αß that was demonstrated by native electrophoresis. Thus, the results indicate a significance of subtype LMP2-PA28αß for tumor and absence of changes in proteasome pool in distal fragments of 2-4 cm from tumor.

Estrogen Receptors and Ubiquitin Proteasome System: Mutual Regulation.

This review provides information on the structure of estrogen receptors (ERs), their localization and functions in mammalian cells. Additionally, the structure of proteasomes and mechanisms of protein ubiquitination and cleavage are described. According to the modern concept, the ubiquitin proteasome system (UPS) is involved in the regulation of the activity of ERs in several ways. First, UPS performs the ubiquitination of ERs with a change in their functional activity. Second, UPS degrades ERs and their transcriptional regulators. Third, UPS affects the expression of ER genes. In addition, the opportunity of the regulation of proteasome functioning by ERs-in particular, the expression of immune proteasomes-is discussed. Understanding the complex mechanisms underlying the regulation of ERs and proteasomes has great prospects for the development of new therapeutic agents that can make a significant contribution to the treatment of diseases associated with the impaired function of these biomolecules.

Combined Effect of Bortezomib and Menadione Sodium Bisulfite on Proteasomes of Tumor Cells: The Dramatic Decrease of Bortezomib Toxicity in a Preclinical Trial.

Tumor growth is associated with elevated proteasome expression and activity. This makes proteasomes a promising target for antitumor drugs. Current antitumor drugs such as bortezomib that inhibit proteasome activity have significant side effects. The purpose of the present study was to develop effective low-toxic antitumor compositions with combined effects on proteasomes. For compositions, we used bortezomib in amounts four and ten times lower than its clinical dose, and chose menadione sodium bisulfite (MSB) as the second component. MSB is known to promote oxidation of NADH, generate superoxide radicals, and as a result damage proteasome function in cells that ensure the relevance of MSB use for the composition development. The proteasome pool was investigated by the original native gel electrophoresis method, proteasome chymotrypsin-like activity-by Suc-LLVY-AMC-hydrolysis. For the compositions, we detected 10 and 20 µM MSB doses showing stronger proteasome-suppressing and cytotoxic in cellulo effects on malignant cells than on normal ones. MSB indirectly suppressed 26S-proteasome activity in cellulo, but not in vitro. At the same time, MSB together with bortezomib displayed synergetic action on the activity of all proteasome forms in vitro as well as synergetic antitumor effects in cellulo. These findings determine the properties of the developed compositions in vivo: antitumor efficiency, higher (against hepatocellular carcinoma and mammary adenocarcinoma) or comparable to bortezomib (against Lewis lung carcinoma), and drastically reduced toxicity (LD50) relative to bortezomib. Thus, the developed compositions represent a novel generation of bortezomib-based anticancer drugs combining high efficiency, low general toxicity, and a potentially expanded range of target tumors.

Interplay between recombinant Hsp70 and proteasomes: proteasome activity modulation and ubiquitin-independent cleavage of Hsp70.

The heat shock protein 70 (Hsp70, human HSPA1A) plays indispensable roles in cellular stress responses and protein quality control (PQC). In the framework of PQC, it cooperates with the ubiquitin-proteasome system (UPS) to clear damaged and dysfunctional proteins in the cell. Moreover, Hsp70 itself is rapidly degraded following the recovery from stress. It was demonstrated that its fast turnover is mediated via ubiquitination and subsequent degradation by the 26S proteasome. At the same time, the effect of Hsp70 on the functional state of proteasomes has been insufficiently investigated. Here, we characterized the direct effect of recombinant Hsp70 on the activity of 20S and 26S proteasomes and studied Hsp70 degradation by the 20S proteasome in vitro. We have shown that the activity of purified 20S proteasomes is decreased following incubation with recombinant human Hsp70. On the other hand, high concentrations of Hsp70 activated 26S proteasomes. Finally, we obtained evidence that in addition to previously reported ubiquitin-dependent degradation, Hsp70 could be cleaved independent of ubiquitination by the 20S proteasome. The results obtained reveal novel aspects of the interplay between Hsp70 and proteasomes.

Amyloid-ß Increases Activity of Proteasomes Capped with 19S and 11S Regulators.

Accumulation of amyloid-ß (Aß) in neurons accompanies Alzheimer's disease progression. In the cytoplasm Aß influences activity of proteasomes, the multisubunit protein complexes that hydrolyze the majority of intracellular proteins. However, the manner in which Aß affects the proteolytic activity of proteasomes has not been established. In this study the effect of Aß42 and Aß42 with isomerized Asp7 on activity of different forms of proteasomes has been analyzed. It has been shown that Aß peptides efficiently reduce activity of the 20S proteasomes, but increase activity of the 20S proteasomes capped with the 19S and/or 11S regulators. Modulation of proteasome activity is mainly determined by the C-terminal segment of Aß (amino acids 17-42). This study demonstrated an important role of proteasome regulators in the interplay between Aß and the proteasomes.

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.

New approach to study of T cellular immunity development: parallel investigation of lymphoid organ formation and changes in immune proteasome amount in rat early ontogenesis.

The expression pattern and distribution of proteasome immune subunits LMP7 and LMP2 in the developing rat spleen and liver as well as the periarterial lymphoid sheath formation were investigated. LMP7 and LMP2 were detected by immunoblotting in the spleen on the 21st embryonic day and during the first postnatal days in equal amounts. Their levels increased by the 8th and 18th postnatal days. Double immunofluorescent labeling the spleen cells revealed LMP7 and LMP2 in T and B lymphocytes localized in the red pulp in embryogenesis. Few T lymphocytes were discovered in periarterial zones on the 8th postnatal day. T lymphocytes filled these zones and formed lymphoid sheaths by the 18-19th day. In the liver, LMP7 and LMP2 were revealed by the 17-19th postnatal day. Immunofluorescent analysis showed their presence in hepatocytes at this period. The data suggest that T cell-mediated immune response in relation to hepatocytes is possible beginning from 18th to 19th postnatal day.