Products of Lipid Peroxidation Reduce Lysosomal Cathepsin Activities in Human Retinal Pigment Epithelial Cells

Abstract

Abstract: : Purpose: A hallmark of early–stage age–related macular degeneration (AMD) is the progressive accumulation of abnormal material in the retinal pigment epithelium (RPE), finally forming lipofuscin granules and drusen. These intra– and extracellular deposits are thought to result from incompletely digested material secondary to impaired lysosomal capacity. In the macula, constant UV–light exposure, along with high oxygen levels, triggers permanent peroxidation of lipids in the photoreceptor membranes. Products of lipid peroxidation like 4–hydroxynonenal (HNE) and malondialdehyde (MDA) are able to bind covalently to proteins, resulting in impaired protein function. Here we study the effect of HNE and MDA on RPE lysosomal enzyme activities. Methods:Pure lysosomal fractions isolated from cultured primary human RPE cells were incubated with either HNE and MDA or isolated bovine photoreceptor outer segments (POS) modified with HNE and MDA. Furthermore, primary human RPE cells and cells of the human RPE cell line ARPE–19 were treated with HNE and MDA. Subsequently, activities of the lysosomal proteases cathepsin B, L, and H were analyzed in isolated lysosomes as well as cultured cells. Results:Both HNE and MDA had a striking effect on cathepsin activities. Treatment of isolated RPE lysosomes with concentrations of 2.5 µM HNE or MDA resulted in a inhibition of cathepsin B and L activities by more than 95%, with the effect of HNE being significantly stronger than MDA. Interestingly, no effect was seen for neither HNE nor MDA on cathepsin H activity. Similarly, incubation of RPE lysosomes with HNE– or MDA–modified POS strongly reduced cathepsin activities. Finally, treatment of both primary RPE cells and ARPE–19 cells with HNE or MDA likewise resulted in a significant reduction of cathepsin activities in these cells. Conclusions: Our results indicate that products of lipid peroxidation may impair lysosomal functions by two kinds of action. (I) HNE and MDA exert an direct inhibitory effect on lysosomal proteolytic activities by covalently binding to the active center of thiolproteases. (II) HNE– and MDA–modified substrate proteins of lysosomal proteases become stabilized against proteolytic attack, thereby converting them to competitive inhibitors of cathepsin activities. Both mechanisms may contribute to RPE dysfunction in early–stage AMD.

Keywords: age-related macular degeneration • retinal pigment epithelium • retinal degenerations: cell biology