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T.U. Krohne, E. Kammerer, J. Kopitz, F.G. Holz; Detection of HNE– and MDA–Modifications on Lysosomal Cathepsins in Human Retinal Pigment Epithelial Cells . Invest. Ophthalmol. Vis. Sci. 2006;47(13):1374.
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Progressive formation of lipofuscin granules and drusen in early–stage age–related macular degeneration (AMD) is thought to result from accumulation of incompletely digested material due to impaired lysosomal function of the retinal pigment epithelium (RPE). Products of lipidperoxidation, in particular 4–hydroxynonenal (HNE) and malondialdehyde (MDA) were previously shown to inactivate lysosomal proteolytic activities in RPE cells. Besides substrate stabilization, covalent binding of MDA and HNE to lysosomal cathepsins is considered a major cause. We tested this hypothesis by directly detecting the corresponding adducts on cathepsin B and L in MDA– and HNE–treated RPE cells.
Cultured human RPE cells were treated with HNE and MDA. Subsequently lysosomes were isolated from the cells and lysosomal proteins separated by 2D–gelelectrophoresis. Protein spots were visualized by fluorescent protein staining. Cathepsin B and L as well as HNE– and MDA–modified proteins were also detected by Western blotting. Matching of the Western Blots to the protein gel finally resulted in the identification of spots representing HNE– and MDA–modified cathepsin B and L, which were further characterized by mass spectrometry.
In HNE– and MDA–treated RPE cells, modifications by these lipidperoxidation products were detected on cathepsin B and L. Mass spectrometric analysis of tryptic peptides obtained from the cathepsins indicated modifications of critical active site residues in both proteases.
Products of lipidperoxidation like HNE and MDA cause covalent modifications in the active center of RPE lysosomal cathepsins. This may result in the observed inhibitory effect of lipidperoxidation products on lysosomal proteolytic activities and may contribute to RPE lysosomal dysfuntion in early–stage AMD.
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