Protein Quality Control by the Ubiquitin Proteolytic Pathway: Roles in Resistance to Oxidative Stress and Disease

There is now consensus that the accumulation of oxidatively modified proteins is cytotoxic and causally related to several age-related diseases including the amyloid diseases and age-related cataracts. There is also general agreement that the ubiquitin proteolytic pathway (UPP) provides a quality control mechanism to limit accumulation of modified proteins. We asked if and how oxidative stress is related to the function of the ubiquitin proteolytic pathway, and vice versa, with the objective of obtaining information that can lead to the development of strategies to delay age-related "amyloid" or "protein precipitation" diseases such as cataracts and age-related macular degeneration. Elevated levels of ubiquitin conjugates were observed when human, rabbit, bovine, and rat lens, retina, liver cells or tissues were exposed to mild oxidative stress, which was created by exposure to paraquat, diamide, peroxide, light together with lipofuscin, and radiomimetic drugs. The increase in ubiquitin conjugates derived from an increase in substrates as well as by hyperactivation of El, rather than inactivation of the proteasome. Using a novel glutathiolated substrate, yC-crystallin, we demonstrated that the UPP shows a previously unrecognized selectivity for such specifically oxidatively modified proteins. Selectivity of the pathway for other oxidatively modified proteins, specifically for protein carbonyls, was indicated in assays that employed the ubiquitin conjugation-competent, but degradation-resistant ubiquitin variant K6W-ubiquitin. These experiments showed that failure to execute ubiquitin-dependent proteolysis renders cells more susceptible to oxidative-stress-related cytotoxicity. Activity of the pathway is regulated in part by cellular redox status, specifically as affected by GSSG. Ubiquitination is enhanced when GSSG/GSH ratios are 0.02-0.15. Since there is potentiation of ubiquitination even when GSSG/GSH ratios are indistinguishable from basal levels, it appears that ubiquitination provides one of the most sensitive indicators of oxidative stress. Ubiquitination is attenuated when GSSG/GSH rises >0.2 and does not occur when GSSG/GSH ≥ 2.9. The data indicate that inhibition of the pathway, which occurs upon aging, is associated with accumulation rather than the timely degradation of ubiquitin conjugates. They further suggest that if the system fails to keep up with production of substrates, high mass ubiquitin conjugates may accumulate and precipitate in cytotoxic aggregates such as are seen in many age-related syndromes, including lens cataracts or in lipofuscin and drusen in the aging retina.