ALDH3A1 Protects Against UVB–Induced Inhibition of the Proteasome

Abstract

Abstract: : Purpose: ALDH3A1 is one of the most abundantly expressed proteins in the mammalian cornea, representing up to 40% of the total water–soluble fraction in some species. Though the precise biological role of ALDH3A1 in the cornea remains to be elucidated, it is clear that ALDH3A1 plays a dynamic role in protecting the cornea against oxidative stress. We have recently demonstrated that cells transfected with human ALDH3A1 were resistant to UV– and 4–hydroxynonenal–induced oxidative damage. In this study, we sought to further characterize the effects of UV radiation by evaluating the activity of the proteasome, which is susceptible to UV–induced inhibition. We hypothesize that ALDH3A1 may prevent UV–induced proteasome damage. Methods: A rabbit keratinocyte cell line (TRK43) was transfected with human ALDH3A1. Control TRK43 cells (empty vector) and ALDH3A1–transfected cells were exposed to UVB–light at doses of 0.05 and 0.25 J/cm². After 24 hr post–exposure incubation, the cells were lysed and assayed for proteosome activity using a substrate for chymotrypsin–like activity (LLVY–AMC). Identical samples were prepared with a proteosome inhibitor (MG 132) to verify the specificity of the assay. In addition, Western blot analysis was used to detect 4–HNE–adducted proteins. Results: In the TRK43 vector cells, we observed that the chymotrypsin–like activity of the proteasome decreased by nearly 60% after both 0.05 and 0.25 J/cm² UVB doses. Cells transfected with ALDH3A1, however, demonstrated no loss in proteasome activity. We did not observe an accumulation of 4–HNE–adduct proteins 24 hours post–exposure in either vector or ALDH3A1–transfected TRK43 cells. Conclusions: In this report, we describe for the first time that ALDH3A1 protects the proteasome against UV–induced inhibition. These findings support the multifunctional role of ALDH3A1 in the protection of the cornea against oxidative stress.

Keywords: cornea: stroma and keratocytes • oxidation/oxidative or free radical damage • protein modifications-post translational