Detection of active proteasome structures in brain extracts: proteasome features of August rat brain with violations in monoamine metabolism

Abstract

The aim of this work was to detect changes in proteasome pools of brain parts of August rats with monoamine metabolism violations in comparison with that of control Wistar rats. To reveal active proteasome structures, a method of native electrophoresis for the analysis of crude tissue fractions was developed. By means of this method and following Western blotting, the most pronounced changes in reorganization of proteasome structures were detected in proteasome pool of the brain cortex of August rats. Main findings are the enhanced expression of immune proteasome subtypes containing proteolytic subunit LMP2 and activator PA28αβ as well as immune proteasome subtypes containing proteolytic subunit LMP7 and activator PA700 and simultaneously decreased expression of subtypes with subunit LMP2 and activator PA700 in the brain cortex of August rats compared to that of Wistar rats. These results were indirectly confirmed by SDS PAGE method followed by Western blotting, which showed the increased quantities of immune subunits and proteasome activators in the brain cortex of August rats compared to that of Wistar rats. Immune proteasomes were revealed by immunohistochemistry in neurons, but not in glial cells of August and Wistar rat cortex. The detected reorganization of proteasome pools is likely to be important for production of special peptides to provide the steady interaction between neurons and adaptation of central nervous system to conditions caused by monoamine metabolism deviations.

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

“…According to native gel electrophoresis method, MSB and bortezomib mutually influenced all proteasome forms in vitro. In particular, cleared homogenates of Hepa 1-6 cells contained three proteasome forms ( Figure 6 A,C): 26S, 20S, and the third form whose position coincided with that of 20S-proteasome bound to 11S-activator (PA28-activator) [ 25 ]. This form utilizes rather short substrates, generates regulatory peptides and functions as a regulatory factor in the cell [ 1 ].…”

“…Fractions enriched in 26S- and 20S-proteasomes were obtained by ammonium sulfate precipitation (0–40% and 40–70% fractions, respectively) [ 26 ]. In addition, proteasome pool of tumor cell cleared homogenates was divided into 26S- and 20S-proteasomes by native 4–10% PAGE developed for crude proteasome fractions [ 25 ].…”

“…Proteasome ChTL-activity was determined by hydrolysis of fluorogenic Suc-LLVY-AMC (Sigma-Aldrich, St. Louis, MO, USA) in 0.5, 1.0, 1.5, and 2.0 μL of the proteasome fraction as described previously [ 25 ]. ChTL-activity was expressed as the substrate quantity (μmol of Suc-LLVY-AMC) hydrolyzed at 37 °C for 20 min and normalized to 10 6 cells or 1000 embryos.…”

“…ChTL-activity was expressed as the substrate quantity (μmol of Suc-LLVY-AMC) hydrolyzed at 37 °C for 20 min and normalized to 10 6 cells or 1000 embryos. Under indicated conditions, the quantity of the reaction product is proportional to reaction duration [ 25 ].…”

“…Proteasome ChTL-activity in native 4–10% polyacrylamide gel was detected with the use of 300 μM Suc-LLVY-AMC (1/20 of gradient gel volume) as described previously [ 25 ]. Fluorescence bands in the gel were photographed under 365 nm UV light.…”