Removal of oxidatively damaged proteins from lens cells by the ubiquitin-proteasome pathway

Understanding how oxidized proteins are removed is important since accumula tion of such damaged proteins is causally related to cellular and organismi c 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 durin g recovery from oxidative stress has a role in selective removal of oxidize d proteins from the cells. In cells which were not exposed to peroxide, inh ibition of the proteasome with MG132 or clasto-lactacystin beta -lactone ha d little effect on protein carbonyl levels. However, inhibition of the prot easome in the 20 muM peroxide-treated cells caused an approximate 60 % incr ease in levels of protein carbonyl and an approximate 100% increase in leve ls of ubiquitin conjugates. The carbonyl-containing proteins that accumulat ed in the presence of the proteasome inhibitor co-localized with high molec ular mass ubiquitin-protein conjugates. Furthermore, isolated carbonyl-cont aining proteins from H2O2-treated cells were ubiquitinated, and ubiquitin-c onjugates were enriched with carbonyl-containing proteins. The diminished e ffect of proteasome inhibitors on protein carbonyl levels, together with th e robust increase in ubiquitin-protein conjugates and accompanied increases in oxidized proteins, upon exposure to 60 muM H2O2 indicate that the prote asomal step of the UPP is more susceptible to oxidative inactivation than t he ubiquitination step. In fact, oxidative stress is associated with a hype ractivation of the ubiquitin-activating enzyme. These data indicate that th e UPP plays a role in removal of oxidatively damaged proteins from cells an d that attenuation of the WP activity may result in cytotoxic accumulation of damaged proteins, possibly including the ubiquitinated forms. (C) 2001 A cademic Press.