Impairment of proteasome function upon UVA- and UVB-irradiation of human keratinocytes

The major environmental influence for epidermal cells is sun exposure and the harmful effect of UV radiation on skin is related to the generation of reactive oxygen species that are altering cellular components including proteins. It is now well established that the proteasome is responsible for the degradation of oxidized proteins. Therefore, the effects of UV-irradiation on proteasome have been investigated in human keratinocyte cultures. Human keratinocytes were irradiated with 10 J/cm2 of UVA and 0.05 J/cm2 of UVB and proteasome peptidase activities were measured in cell lysates using fluorogenic peptides. All three peptidase activities were decreased as early as 1 h and up to 24 h after irradiation of the cells. Increased levels of oxidized and ubiquitinated proteins as well as proteins modified by the lipid peroxidation product 4-hydroxy-2-nonenal were also observed in irradiated cells. However, immunopurified 20S proteasome exhibited no difference in both peptidase specific activities and 2D gel pattern of subunits in irradiated cells, ruling out the possibility that the 20S proteasome could be a target for the UV-induced damage. Finally, extracts from irradiated keratinocytes were able to inhibit degradation by the proteasome, demonstrating the presence of endogeneous inhibitors, including 4-hydroxy-2-nonenal modified proteins, generated upon UV-irradiation.