Removal of oxidatively damaged proteins from lens cells by the ubiquitin-proteasome pathway.
Understanding how oxidized proteins are removed is important since accumulation of such damaged proteins is causally related to cellular and organismic dysfunction, disease and aging. Previous work showed that activity of the ubiquitin-proteasome pathway (UPP) in lens cells increased during recovery from oxidative stress ( Shang et al., 1997b : J. Biol. Chem. 272, 23086-93). In this study we sought to determine if the up-regulation of the UPP during recovery from oxidative stress has a role in selective removal of oxidized proteins from the cells. In cells which were not exposed to peroxide, inhibition of the proteasome with MG132 or clasto-lactacystin beta-lactone had little effect on protein carbonyl levels. However, inhibition of the proteasome in the 20 microM peroxide-treated cells caused an approximate 60% increase in levels of protein carbonyl and an approximate 100% increase in levels of ubiquitin conjugates. The carbonyl-containing proteins that accumulated in the presence of the proteasome inhibitor co-localized with high molecular mass ubiquitin-protein conjugates. Furthermore, isolated carbonyl-containing proteins from H2O2-treated cells were ubiquitinated, and ubiquitin-conjugates were enriched with carbonyl-containing proteins. The diminished effect of proteasome inhibitors on protein carbonyl levels, together with the robust increase in ubiquitin-protein conjugates and accompanied increases in oxidized proteins, upon exposure to 60 microM H2O2 indicate that the proteasomal step of the UPP is more susceptible to oxidative inactivation than the ubiquitination step. In fact, oxidative stress is associated with a hyperactivation of the ubiquitin-activating enzyme. These data indicate that the UPP plays a role in removal of oxidatively damaged proteins from cells and that attenuation of the UPP activity may result in cytotoxic accumulation of damaged proteins, possibly including the ubiquitinated forms.
Shang
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Laboratory for Nutrition and Vision Research, Jean Mayer USDA Human Nutrition Research Center on Aging at Tufts University, 711 Washington Street, Boston, MA 02111, USA.
Nowell
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TR
Taylor
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A
D000818:Animals; D003459:Crystallins / Q000378:metabolism*; D015853:Cysteine Proteinase Inhibitors / Q000494:pharmacology; D007783:Lactones / Q000494:pharmacology; D007908:Lens, Crystalline / Q000166:cytology / Q000502:physiology*; D007976:Leupeptins / Q000494:pharmacology; D008565:Membrane Proteins / Q000378:metabolism; D008970:Molecular Weight; D018384:Oxidative Stress / Q000502:physiology*; D010447:Peptide Hydrolases / Q000378:metabolism*; D011817:Rabbits; D014452:Ubiquitins / Q000378:metabolism*; D015854:Up-Regulation
D003459:Crystallins; D015853:Cysteine Proteinase Inhibitors; D007783:Lactones; D007976:Leupeptins; D008565:Membrane Proteins; D014452:Ubiquitins; C098869:clasto-lactacystin beta-lactone; D010447:Peptide Hydrolases; C072553:benzyloxycarbonylleucyl-leucyl-leucine aldehyde