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 H(2)O(2) 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 H(2)O(2) concentrations of up to 5 mM, the ATP-stimulated degradation of suc-LLVY-MCA by the 26S proteasome began to decline at 400 microM 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 H(2)O(2) but not after 2 mM H(2)O(2). 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.
Reinheckel
T
T
Clinics of Physical Medicine and Rehabilitation, Medical Faculty (Charité), Humboldt University Berlin, Schumannstrasse 20/21, Berlin, D-10098, Germany.
Ullrich
O
O
Sitte
N
N
Grune
T
T
D000111:Acetylcysteine / Q000031:analogs & derivatives / Q000494:pharmacology; D000255:Adenosine Triphosphate / Q000494:pharmacology; D002470:Cell Survival / Q000187:drug effects; D003374:Coumarins / Q000378:metabolism; D003546:Cysteine Endopeptidases / Q000378:metabolism*; D015853:Cysteine Proteinase Inhibitors / Q000494:pharmacology; D004305:Dose-Response Relationship, Drug; D005456:Fluorescent Dyes / Q000378:metabolism; D006801:Humans; D006861:Hydrogen Peroxide / Q000494:pharmacology; D020014:K562 Cells; D009097:Multienzyme Complexes / Q000378:metabolism*; D009842:Oligopeptides / Q000378:metabolism; D016877:Oxidants / Q000494:pharmacology; D010084:Oxidation-Reduction / Q000187:drug effects; D018384:Oxidative Stress / Q000187:drug effects; D010447:Peptide Hydrolases / Q000378:metabolism*; D046988:Proteasome Endopeptidase Complex; D013997:Time Factors
D003374:Coumarins; D015853:Cysteine Proteinase Inhibitors; D005456:Fluorescent Dyes; D009097:Multienzyme Complexes; D009842:Oligopeptides; D016877:Oxidants; C067713:lactacystin; D000255:Adenosine Triphosphate; C046582:succinyl-leucyl-leucyl-valyl-tyrosyl-methylcoumarinamide; D006861:Hydrogen Peroxide; D010447:Peptide Hydrolases; D003546:Cysteine Endopeptidases; D046988:Proteasome Endopeptidase Complex; C061553:ATP dependent 26S protease; D000111:Acetylcysteine