Upon oxidative stress cells show an increase in the oxidized glutathione (GSSG) to reduced glutathione (GSH) ratio with a concomitant decrease in activity of the ubiquitinylation pathway. Because most of the enzymes involved in the attachment of ubiquitin to substrate proteins contain active site sulfhydryls that might be covalently modified (thiolated) upon enhancement of GSSG levels (glutathiolation), it appeared plausible that glutathiolation might alter ubiquitinylation rates upon cellular oxidative stress. This hypothesis was explored using intact retina and retinal pigment epithelial (RPE) cell models. Exposure of intact bovine retina and RPE cells to H2O2 (0.1–1.7 μmol/mg) resulted in a dose-dependent increase in the GSSG:GSH ratio and coincident dose-dependent reductions in the levels of endogenous ubiquitin-activating enzyme (E1)-ubiquitin thiol esters and endogenous protein-ubiquitin conjugates and in the ability to formde novo retinal protein-125I-labeled ubiquitin conjugates. Oxidant-induced decrements in ubiquitin conjugates were associated with 60–80% reductions in E1 and ubiquitin-conjugating enzyme (E2) activities as measured by formation of ubiquitin thiol esters. When GSH levels in RPE cells recovered to preoxidation levels following H2O2 removal, endogenous E1 activity and protein-ubiquitin conjugates were restored. Evidence that S thiolation of E1 and E2 enzymes is the biochemical link between cellular redox state and E1/E2 activities includes: (i) 5-fold increases in levels of immunoprecipitable, dithiothreitol-labile35S-E1 adducts in metabolically labeled, H2O2-treated, RPE cells; (ii) diminished formation of E1- and E2-125I-labeled ubiquitin thiol esters, oligomerization of E225K, and coincident reductions in protein-125I-labeled ubiquitin conjugates in supernatants from nonstressed retinas upon addition of levels of GSSG equivalent to levels measured in oxidatively stressed retinas; and (iii) partial restoration of E1 and E2 activities and levels of protein-125I-labeled ubiquitin conjugates in supernatants from H2O2-treated retinas when GSSG:GSH ratios were restored to preoxidation levels by the addition of physiological levels of GSH. These data suggest that the cellular redox status modulates protein ubiquitinylation via reversible S thiolation of E1 and E2 enzymes, presumably by glutathione.
Dietary Glycemia Contributes Reversibly to Age-related Macular Degeneration and Metabolic Disease in Aged C57Bl/6J Mice Objective Consuming high glycemia diets is a risk factor for Age-related Macular Degeneration (AMD) in humans. We sought to test in aging C57Bl/6J mice whether a high glycemic index (GI) diet would lead to AMD and other metabolic changes, and if the damage is reversible. Methods: 1-year old mice were fed high or low GI diets for one year. A crossover group had their diets shifted from high to low GI at the mid-point. Retina pathology, metabolic parameters, metabolites, advanced glycation end-products, and microbiota were measured. Results: Mice fed a high GI diet developed obesity and insulin resistance. Many metabolic pathways, including tyrosine, citrate, and ascorbate pathways, and gut microbiomes showed diet-associated changes. High GI-fed mice showed loss of photoreceptor cells and retina pathology consistent with early AMD. Pathology was less prominent in mice in the low GI or crossover groups. Conclusions Chronic consumption of a high glycemia diet induces metabolic disease and AMD, which can be reversed by lowering dietary glycemia, suggesting that diet could be a primary intervention in treatment of early AMD. Funded by NIH (RO1EY012121, RO1EY13250) and USDA (1950-510000-060-01A)
In the aged lens Dostsynthetically altered molecules comprise the majority of lens proteins. Many proteolytic activities have been observed in lens suoernatants. Since damaged or altered proteins are usually selectively and rapidly degraded in other cells and tissues, the accumulation of these soecies in the lens seemed enigmatic. Initiation of Droteolysis has been studied most extensively in reticulocytes and ts 35 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: 1) ubiquitin is Dresent in_the lens; 2) lens Droteins are conjugated to I-ubiquitin using reticulocyte conjugating systems; 3) the reaction is ATP detiendent; 4) proteins from lens epithelium/outer cortex and core form different ubiquitin conjugates; 5) lens proteins compete with lysozyme and reticulocyte nroteins for the ubiquitin conjugating apparatus; 6) most of the conjugates are of very high molecular weight; 7) there is a temporal nature to the Dattern of conjugates observed; and 8) the ubiquitin conjugation system shows extreme selectivity.
Although there is ample evidence that the advanced glycation end-product (AGE) glucosepane contributes to age-related morbidities and diabetic complications, the impact of glucosepane modifications on proteins has not been extensively explored due to the lack of sufficient analytical tools. Here, we report the development of the first polyclonal anti-glucosepane antibodies using a synthetic immunogen that contains the core bicyclic ring structure of glucosepane. We investigate the recognition properties of these antibodies through ELISAs involving an array of synthetic AGE derivatives and determine them to be both high-affinity and selective in binding glucosepane. We then employ these antibodies to image glucosepane in aging mouse retinae via immunohistochemistry. Our studies demonstrate for the first time accumulation of glucosepane within the retinal pigment epithelium, Bruch's membrane, and choroid: all regions of the eye impacted by age-related macular degeneration. Co-localization studies further suggest that glucosepane colocalizes with lipofuscin, which has previously been associated with lysosomal dysfunction and has been implicated in the development of age-related macular degeneration, among other diseases. We believe that the anti-glucosepane antibodies described in this study will prove highly useful for examining the role of glycation in human health and disease.
