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F.G. Holz, E. Kämmerer, A. Bindewald, J. Kopitz; Effects of Lipidperoxidation–related Protein Modifications on RPE Lysosomal Functions, ROS Phagocytosis and their Impact for Lipofuscinogenesis . Invest. Ophthalmol. Vis. Sci. 2004;45(13):3385.
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© ARVO (1962-2015); The Authors (2016-present)
Purpose: Lipofuscin (LF) accumulation in the RPE is a common downstream pathogenetic pathway in various monogenetic and complex retinal diseases including age–related macular degeneration (AMD). Lipid peroxidation inducing modifications of proteins is thought to play a role in lipofuscinogenesis and may contribute to RPE dysfunction. We previously demonstrated that a variety of LF–proteins are damaged by aberrant covalent modifications of malondialdehyde (MDA), 4–hydroxynonenal (HNE) and AGE. Here we tested the hypothesises that these damaged proteins are more resistant to proteolytic attack, act as protease inhibitors and affect ROS phagocytosis. Methods: We modified in vitro isolated ROS and endogenous RPE proteins with MDA and HNE, and examined pure lysosomal fractions isolated from human RPE cultures for their capability to degrade modified proteins. In parallel, modified and radiolabelled ROS were fed to RPE cell cultures for phagocytosis and their lysosomal degradation was compared to unmodified ROS. Results: Both experimental approaches revealed that MDA– or HNE–modifications strikingly reduce the degradation of phagocytosed ROS or autophagocytosed cytoplasmic proteins. Degradation rates of MDA– or HNE–modified ROS were reduced by 52% and 61%, respectively, when measured with isolated lysosomal fractions and by 64% and 51%, respectively, in the cell culture as compared to unmodified ROS. Some of the modified proteins remained undegraded in the lysosomal compartment of cultured RPE cells and were detectable more than 3 weeks after feeding, whereas unmodified ROS were degraded with a halflife of about 24h with no detectable remnants 1 week after feeding. Modified proteins had the potential to impair degradation of unmodified proteins. The presence of an excess of modified proteins reduced degradation rates for unmodified ROS up to 70% indicating a competitive inhibitory action on normal function of lysosomal proteases. This competitive effect was confirmed in experiments in which activities of the lysosomal cathepsins B, D and L were reduced by more than 80% in the presence of MDA– or HNE–modified proteins as compared to unmodified controls. Conclusions: Our results indicate that lipid peroxidation–derived protein modifications are involved in lipofuscinogenesis and may contribute to cell–damaging effects of LF in retinal diseases associated with excessive LF such as AMD.
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