Proteasome-dependent turnover of protein disulfide isomerase in oxidatively stressed cells.
Generalized increases in protein oxidation and protein degradation in response to mild oxidative stress have been widely reported, but only a few individual proteins have actually been shown to undergo selective, oxidation-induced proteolysis. Our goal was to find such proteins in Clone 9 liver cells exposed to hydrogen peroxide. Using metabolic radiolabeling of intracellular proteins with [35S]cysteine/methionine, and analysis by two-dimensional polyacrylamide gel electrophoresis (2-D PAGE), we found at least three labeled proteins ("A," "B," and "C") whose levels were decreased significantly more than the generalized protein loss after mild oxidative stress. "Protein C" was excised from 2-D PAGE and subjected to N-terminal amino acid microsequencing. "Protein C" was identified as Protein Disulfide Isomerase or PDI (E.C. 5.3.4.1), and this identity was reconfirmed by Western blotting with a C-terminal anti-PDI monoclonal antibody. A combination of quantitative radiometry and Western blotting in 2-D PAGE revealed that PDI was selectively degraded and then new PDI was synthesized, following H2O2 exposure. PDI degradation was blocked by inhibitors of the proteasome, and by cell treatment with proteasome C2 subunit antisense oligonucleotides, indicating that the proteasome was largely responsible for oxidation-induced PDI degradation.
Grune
Tilman
T
Ethel Percy Andrus Gerontology Center, the University of Southern California, Los Angeles, California 90089-0191, USA.
Reinheckel
Thomas
T
Li
Rui
R
North
James A
JA
Davies
Kelvin J A
KJ
D000818:Animals; D002478:Cells, Cultured; D003546:Cysteine Endopeptidases / Q000378:metabolism*; D004305:Dose-Response Relationship, Drug; D004847:Epithelial Cells / Q000166:cytology; D006861:Hydrogen Peroxide / Q000494:pharmacology; D008099:Liver / Q000166:cytology; D009097:Multienzyme Complexes / Q000037:antagonists & inhibitors / Q000378:metabolism*; D018384:Oxidative Stress / Q000502:physiology*; D046988:Proteasome Endopeptidase Complex; D019704:Protein Disulfide-Isomerases / Q000378:metabolism*; D051381:Rats
D009097:Multienzyme Complexes; D006861:Hydrogen Peroxide; D003546:Cysteine Endopeptidases; D046988:Proteasome Endopeptidase Complex; D019704:Protein Disulfide-Isomerases