ИСТИНА

Proteasome functions in every eukaryotic cell, degrading most of the irregular or nonviable cellular proteins. Turning off proteasome leads to disastrous consequences, and finally to cell death. The polyglutamine (polyQ) repeat diseases are inherited neurodegenerative disorders attributed to elongation of polyQ fragments (from 6–35 to 36–306 glutamine residues) in certain proteins. These abnormal proteins form aggregates and become insoluble, resulting in cytoplasmic or nuclear protein inclusions in some brain regions. Analysis of inclusions reveals presence of mutant proteins, and also of ubiquitin and components of 26S proteasome (20S core, 19S and 11S regulatory particles). It is assumed that proteasomal degradation system attempts to digest abnormal misfolded proteins, but fails to cut within polyQ regions or does it with low efficiency. Then proteasome may become clogged and inactivated by these long aggregated polyQ sequences. The aim of our study was to investigate the effect of the 11S regulator on proteasomal degradation of polyQ-containing protein, namely huntingtin (htt). 20S, 26S proteasome and 11S regulator were isolated from mice brains. Homogenates were subjected to stepwise salt fractionation with subsequent purification by different chromatographic methods (gel filtration and anion exchange chromatography). Isolated proteins were characterized by molecular weight using gel-filtration and Western-blot. The dependency of 20S proteasome peptidase activity on concentration of 11S regulator was examined. Kinetic constants of proteasome hydrolysis of three peptide substrates (Suc-LLVY-AMC, Z-LLE-AMC and Ac-RLR-AMC) were studied in presence of 11S regulator. HEK cells were transfected by vector containing 15Q or 138Q htt gene (generous gift of Prof. E.V. Kaznacheeva, St.-Petersburgh) and cell lysates were subjected to hydrolysis by 20S proteasome with or without 11S regulator. Reactions were followed by Westernblot (anti-htt antibodies were kindly provided by Vladimir A. Vigont). Our data suggest a possible acceleration of proteasomal degradation of htt in the presence of the 11S regulator.