Purpose: Lipofuscin accumulation in the RPE is a common downstream pathogenic pathway in various monogenic and complex retinal diseases including age-related macular degeneration (AMD). Lipid peroxidation-induced modification of proteins is thought to play a role in lipofuscinogenesis and may contribute to RPE dysfunction. A prior study demonstrated that a variety of lipofuscin-associated proteins are damaged by aberrant covalent modifications of malondialdehyde (MDA) and 4-hydroxynonenal (HNE). The present study was conducted to test the hypothesis that these damaged proteins are more resistant to proteolytic attack and act as protease inhibitors.
Methods: Isolated photoreceptor outer segments (POS) were radioactively labeled and in vitro modified with MDA and HNE. Pure lysosomal fractions isolated from human RPE were tested for their proteolytic activities toward modified and unmodified POS proteins. In parallel, modified and radiolabeled POS were fed to RPE cell cultures for phagocytosis and their lysosomal degradation as well as intracellular accumulation was compared with unmodified POS.
Results: Both experimental approaches revealed that MDA or HNE modifications strikingly increase the resistance of POS proteins to the attack by lysosomal proteases. When cultured RPE cells were fed with modified or unmodified POS the amount of degraded POS proteins was reduced by approximately 60% to 70% for the modified POS compared with those in normal control subjects. Some of the modified proteins remained undegraded in the lysosomal compartment of cultured RPE cells and were still detectable 3 weeks after feeding, whereas unmodified POS were completely degraded within 1 week after feeding. Moreover, modified proteins had the potential to impair degradation of unmodified proteins, indicating their efficacy as proteolytic antagonists.
Conclusions: The results indicate that lipid peroxidation-derived protein modifications are involved in lipofuscinogenesis and may contribute to cell damaging effects of lipofuscin in retinal diseases such as AMD.