AGE-RELATED DECLINE IN UBIQUITIN CONJUGATION IN RESPONSE TO OXIDATIVESTRESS IN THE LENS

Accumulation of damaged proteins is a major age-related change in lens es of virtually all species and is associated with lens opacification. Proteolytic removal of the damaged proteins may play an important rol e in maintaining the transparency of the lens. In many tissues, select ive removal of abnormal or damaged proteins occurs via a ubiquitin-dep endent proteolytic pathway. Ubiquitin, an 8.5 kDa polypeptide, selecti vely binds to proteins to form ubiquitin-protein conjugates. This ubiq uitin-protein conjugate is, in most cases, a signal for protein degrad ation. In this work, age-related changes in rat lens in the following aspects were detected: (a) levels of the ubiquitin-protein conjugates, (b) some of the enzymes involved in ubiquitin conjugation in rat lens es, and (c) ability to respond to oxidative damage. Endogenous ubiquit in-protein conjugates were detected in epithelium, cortex and nucleus of lenses from young and old rats. The levels of endogenous high molec ular weight (HMW) ubiquitin-protein conjugates in each developmental z one of the lenses from young rats were higher than that in the counter parts of lenses from old animals. Peroxide-treatment generally resulte d in elevated levels of endogenous HMW ubiquitin-protein conjugates al though masses of bulk proteins remain unchanged. The increases in ubiq uitin-protein conjugates in the epithelial sections of young and old l enses upon oxidative stress were comparable. In the cortex of young le nses, there was a significant oxidation-related increase in ubiquitin- protein conjugates. There was a similar trend but diminished response in the cortex of old lenses. Nuclear fibers from young lenses also sho wed an oxidation-induced increase in the level of ubiquitin-protein co njugates. This response was not observed in nuclear fibers of old lens es. The ability to form HMW-ubiquitin conjugates with exogenous I-125- labeled ubiquitin in the lens also increased upon oxidative stress. Th e extent of the increase in the de-novo ubiquitin conjugating activity upon exposure to oxidation in old lens was much smaller than in young lens. Ubiquitin-activating enzyme (E1), and ubiquitin conjugating enz ymes (E2(17k), E2(20k) and E2(25k)) were detected by thiol ester assay s or Western blot analysis, No significant age-related changes in the levels of E1, E2(17k), E2(20k) and E2(25k) were detected. The activity of E1 and E2(17k) increased upon exposure to H2O2. These data indicat e that lens has the ability to increase ubiquitin conjugation activity in response to oxidative stress and this ability is attenuated upon a ging. The age-related decrease in the ability to mount a ubiquitin-dep endent response upon oxidation may contribute to the accumulation of d amaged proteins in the old lenses. (C) 1997 Academic Press Limited