Lens proteins are substrates for the reticulocyte ubiquitin conjugation system

Abstract

In the aged lens postsynthetically altered molecules comprise the majority of lens proteins. Many proteolytic activities have been observed in lens supernatants. Since damaged or altered proteins are usually selectively and rapidly degraded in other cells and tissues, the accumulation of these species in the lens seemed enigmatic. Initiation of proteolysis has been studied most extensively in reticulocytes and ts 85 cells. In these systems proteolysis is absolutely ATP dependent, occurs effectively on high molecular weight substrates and, at least for a majority of proteolytic reactions, requires conjugation of ubiquitin to putative substrates. It seemed plausible that the accumulation of high molecular weight protein aggregates in older lenses might be due to the attenuated function of these ubiquitin- and ATP-dependent components in the initial committing processes of proteolysis. This research shows that: ubiquitin is present in the lens; lens proteins are conjugated to 125I-ubiquitin using reticulocyte conjugating systems; the reaction is ATP dependent; proteins from lens epithelium/outer cortex and core form different ubiquitin conjugates; lens proteins compete with lysozyme and reticulocyte proteins for the ubiquitin conjugating apparatus; most of the conjugates are of very high molecular weight; there is a temporal nature to the pattern of conjugates observed; and the ubiquitin conjugation system shows extreme selectivity.

Activity of Ubiquitin-dependent Pathway in Response to Oxidative Stress

“…We previously hypothesized that one role for the ubiquitin pathway is to remove damaged and cytotoxic proteins (3,40). In support of this hypothesis, data in Figs.…”

“…Shortly after the discovery of the pathway (51,52), it was shown that some oxidized proteins are selectively degraded by this proteolytic pathway (18,53,54). Previously we demonstrated that 1) lens and retina tissues have ubiquitin-dependent proteolytic pathways (19,35,40,50,(55)(56); 2) oxidized lens proteins are selective substrates for the pathway (18); 3) efficiency of the pathway is related to cellular redox status (57); and 4) that lenses and lens epithelial cells in culture mount a robust ubiquitinylation response to oxidative stress (19,35 ubiquitinylation process are up-regulated in response to stress in these various experimental systems. The data in Figs.…”

“…The masses of E2-ubiquitin thiol esters are ϳ8.5 kDa higher than the masses of the corresponding E2s (38). The concentrations of 125 I-ubiquitin in both the thiol ester and conjugation assays were saturating and were 15-30-fold higher than the concentrations of endogenous unlabeled ubiquitin (40). Thus, competition between the endogenous unlabeled ubiquitin and the 125 I-ubiquitin to form thiol esters or conjugates was negligible.…”

Ubiquitin is associated with abnormal cytoplasmic filaments characteristic of neurodegenerative diseases.

“…After heat shock, however, there is an increase in the amount and stability of Tritoninsoluble high molecular weight ubiquitin conjugates, suggesting an association with the cytoskeleton (34). Moreover, the rate ofprotein degradation decreases after heat shock (34,35) and during cellular aging (36,37). The increase in stable ubiquitin conjugates has been tentatively attributed to a saturation of the ubiquitin-mediated proteolytic system by an increased amount of altered proteins, as the ubiquitinating and ubiquitin-dependent proteolytic activities have been found to be maintained (34,38).…”

“…Identification of the ubiquitin-acceptor proteins in these inclusions is likely to provide valuable insight into their mechanism of formation and to reveal the roles of the ubiquitin system in the pathology of the nervous system. which are known to occur with increased frequency during aging (37); it could also be due to sustained cellular stress related to the disease process, or to a combination of these events.…”