Abstract
Abstract: :
Purpose: We have previously reported that proteasome chymotryptic–like activity is decreased in the aged retina (Louie et al, 2002). This age–dependent loss in activity occurred concurrent with an increase in retinal proteins modified by 4–hydroxynonenal (HNE), a product of lipid peroxidation that covalently attaches to proteins. A second reaction between the HNE–adduct and an adjacent lysine residue can generate intra– and intermolecular crosslinks. We asked if HNE modification of select proteasomal subunits could be involved in the catalytic site–specific loss in activity. Methods: Retinal proteasomes immunoprecipitated from young and aged rat retina were probed for HNE–adducts and crosslinks by Western immunoblotting. To directly assess if HNE–modification could inhibit activity, purified 20S proteasome was incubated with HNE. The extent of modification was evaluated by Western immunoblotting. Activity of the three catalytic sites was monitored over time of incubation using fluorogenic peptides. Results: Proteasome immunoprecipitated from retina demonstrated an age–related increase in HNE–modified subunits migrating at the expected masses of 20–30 kDa and also at a higher mass of ∼50–60 kDa. Reprobing membranes with an antibody that recognizes HNE cross links produced an immune reaction that colocalized with the proteasome subunits at ∼50–60 kDa. These results are consistent with direct modification of proteasomal subunits by HNE that can also produce HNE–crosslinked subunits. Incubation of purified 20S proteasome with HNE resulted in a time–dependent increase in HNE–modified and cross linked subunits. With HNE–incubation, activity was inhibited at all three catalytic sites, albeit at different rates and extents. The chymotrypsin–like activity was inhibited 70 times faster than the other two activities. Maximal inhibition was two times greater for both the chymotrypsin– and trypsin–like activities compared with the peptidylglutamyl hydrolyzing activity. Conclusions:This study provides in vivo evidence for HNE modification of retinal 20S proteasome. In addition, the chymotrypsin–like activity was the most sensitive to in vitro inhibition by HNE. These results suggest a role for HNE in age–related changes in retinal proteasome function.
Keywords: oxidation/oxidative or free radical damage • protein modifications–post translational • proteolysis