Proteasome inhibition in glyoxal-treated fibroblasts and resistance of glycated glucose-6-phosphate dehydrogenase to 20 S proteasome degradation in vitro

Glycation and glycoxidation protein products are formed upon binding of sug ars to NH2 groups of lysine and arginine residues and have been shown to ac cumulate during aging and in pathologies such as Alzheimer's disease and di abetes. Because the proteasome is the major intracellular proteolytic syste m involved in the removal of altered proteins, the effect of intracellular glycation on proteasome function has been analyzed in human dermal fibrobla sts subjected to treatment with glyoxal that promotes the formation of N ep silon -carboxymethyl-lysine adducts on proteins. The three proteasome pepti dase activities were decreased in glyoxal-treated cells as compared with co ntrol cells, and glyoxal was also found to inhibit these peptidase activiti es in vitro. In addition, the activity of glucose-6-phosphate dehydrogenase , a crucial enzyme for the regulation of the intracellular redox status, wa s dramatically reduced in glyoxal-treated cells. Further analysis was perfo rmed to determine whether glycated proteins are substrates for proteasome d egradation. In contrast to the oxidized glucose-6-phosphate dehydrogenase, both N epsilon -carboxymethyl-lysine- and fluorescent-glycated enzymes were resistant to degradation by the 20 S proteasome in vitro, and this resista nce was correlated with an increased conformational stability of the glycat ed proteins. These results provide one explanation for why glycated protein s build up both as a function of disease and aging. Finally, N epsilon -car boxymethyl-lysine-modified proteins were found to be ubiquitinated in glyox al-treated cells suggesting a potential mechanism by which these modified p roteins may be marked for degradation.