| Chronic exposure to solar UVA plays a causative role in skin photoaging and photocarcinogenesis. Guided by exploratory difference-in-gel-electrophoresis (DIGE)-proteomics, we identified the thiol-dependent cysteine-proteases cathepsin B and cathepsin L as novel UVA-targets undergoing photo-oxidative inactivation upstream of autophagic-lysosomal dysfunction. In human skin fibroblasts, exposure to noncytotoxic doses of chronic UVA (9.9 J/cm2, twice a week, 3 weeks) caused pronounced photooxidative impairment of cathepsin B and L enzymatic activity suppressed by antioxidant intervention. Western blot analysis revealed extensive 4-hydroxy-2-trans-nonenal (4-HNE) modification of cathepsin B in UVA-exposed fibroblasts. Consistent with lysosomal impairment, accumulation of cellular autofluorescent material colocalizing with lysosomes was observed by confocal fluorescence microscopy, and extensive deposition of lipofuscin was detectable by transmission electron microscopy (TEM). Lysosomal expansion was further evidenced by increased immunodetection of lysosomal associated membrane protein-1 (Lamp-1) and Lysotracker-based flow cytometric analysis. While lysosomal membrane integrity remained intact, autophagic blockade was suggested by accumulation of cellular protein levels of LC3-II and p62 (sequestosome 1) in UVA-exposed fibroblasts. Furthermore, UVA-exposure modulated transcriptional levels of p62 (sequestosome 1, SQSTM1), &... |
