Targeted disruption of specific steps of the ubiquitin-proteasome pathway by oxidation in lens epithelial cells

Several steps in the ubiquitin-proteasome pathway have been shown to be inhibited in models of oxidative stress and aging. We have designed similar models of aging and oxidation in the HLE B-3 human lens epithelial cell line. Following hydrogen peroxide (H2O2) treatment, B-3 cells exhibited an expected activation of c-fos. The effect of these same and similar treatments on the lens proteasome system was unexpected. The 2D gel pattern and the chymotrypsin-like activity of the 20S core were unaffected by this H2O2 treatment, contrary to previous experience in other culture systems. The critical role of proteolysis in the aging lens, and the strong tie between oxidation and proteasome changes, urged us to further model lens oxidation and investigate several steps of the ubiquitin-proteasome pathway with an alternative agent: the thiol-specific oxidant, diamide. The 20S core proteasome, de-ubiquitinating, and ATP-dependent 26S proteasome activities all showed decreases 10 min after diamide was applied, and recovered to near normal within 1 h. The higher, 300 muM dose inhibited the 20S by 43%, the de-ubiquitinating activity by 17% and the 26S by 31%. The comparable susceptibility of the 20S activity and the 26S activity differs from several previously published models. Such differences may be the result of tissue or cell line-specific variants in either the components of the ubiquitin-proteasome pathway or in their modification by intracellular oxidants or reductants. (C) 2003 Elsevier Science Ltd. All rights reserved.