Differential impairment of 20S and 26S proteasome activities in human hematopoietic K562 cells during oxidative stress

The 20S proteasome and the 26S proteasome are major components of the cytosolic and nuclear proteasomal proteolytic systems. Since proteins are known to be highly susceptible targets for reactive oxygen species, the effect of H2O2 treatment of K562 human hematopoietic cells toward the activities of 20S and 26S proteasomes was investigated, While the ATP-independent degradation of the fluorogenic peptide suc-LLVY-MCA was not affected by H2O2 concentrations of up to 5 mM, the ATP-stimulated degradation of suc-LLVY-MCA by the 26S proteasome began to decline at 400 mu M and was completely abolished at 1 mM oxidant treatment. A combination of nondenaturing electrophoresis and Western blotting let us believe that the high oxidant susceptibility of the 26S proteasome is due to oxidation of essential amino acids in the proteasome activator PA 700 which mediates the ATP-dependent proteolysis of the 26S-proteasome. The activity of the 26S-proteasome could be recovered within 24 h after exposure of cells to 1 mM H2O2 but not after 2 mM H2O2. In view of the specific Functions of the 26S proteasome in cell cycle control and other important physiological functions, the consequences of the higher susceptibility of this protease toward oxidative stress needs to be considered. (C) 2000 Academic Press.