Oxidative stress is known to cause a range of types of damage to proteins, including peptide backbone breakage and residue side chain modifications, such as deamination and cross-linkage. Since proteins damaged in this manner often have reduced biological activity and since the total amount of protein which can be dissolved in a cell is limited, the cell needs to be able to eliminate or repair such damaged proteins in order to continue functioning. Since it has previously been shown in vivo that oxidized proteins are more susceptible to degradation by the multicatalytic proteasome and that this represents the major cellular pathway whereby such proteins are degraded, this project has examined whether the activity and subunit composition of the proteasome are altered in response to an oxidative stress by hydrogen peroxide in an HeLa cell culture system. No marked alteration in proteasome composition was detectable. In addition, no alteration in ubiquitin-binding proteins was evident. The rate of protein turnover in HeLa cells exposed to a variety of oxidative stresses increased as compared to control cells. Interestingly, a substantial proportion (40%) of this increase was not inhibitable by the proteasome inhibitors MG132 or lactacystin. This raises the interesting possibility that other proteases may be involved in the removal of oxidatively damaged proteins. It has previously been reported (Gershon, 1983) that antibodies raised against proteins that have been denatured by boiling in 2% SDS/2% mercaptoethanol are able to precipitate denatured forms of those proteins from the cytosol of rats, and that an increasing proportion of such proteins accumulate in the cytosol of aging rat liver. Antibody has been raised against cytosol treated in this manner. Curiously, this antibody was found to be more reactive towards cytosol that had been oxidized by an iron/ascorbate system than the SDS-treated cytosol used to raise it. In addition, preliminary results suggest that cytosol from old rats contains more immunoprecipitatable material than that from younger rats. Work is currently being carried out to characterize the protein immunoprecipitate.
