Abstract
Purpose:
The protein Peroxiredoxin (Prdx) 6 and its transactivator Sp1 are aberrantly Sumoylated by Sumo (Small Ubiquitin-like Modifier)-1, resulting in loss of Prdx6 expression and protective activity and leading to lens epithelial cell (LEC) death. We investigated whether oxidative stress-induced aberrant Sumoylation signaling can be reversed by delivering TAT transduction-linked mutant Prdx6K(Lysine)122/142 R(Arginine) at its Sumoylation sites.
Methods:
Sumoylation status of Prdx6 and Sp1 in LECs or lenses that were aging or facing oxidative stress and their correlation with cell survival were examined by sandwich-Sumo1/Prdx6 specific-ELISA, immunoprecipitation, Western blot and qPCR using their corresponding probes. MTS, TUNEL and H2DCFH-DA dye assays examined cell viability and measured reactive oxygen levels, respectively. Prdx6-deficent LECs overexpressing Sumo1 were generated by transfecting pGFP-Sumo1. Prdx6 cDNA and its mutant at Sumo-1, K122/142 to R (by Site-Directed Mutagenesis) were cloned into pTAT-HA prokaryotic expression vector, and recombinant wild type (wt) and Mutant (mut) Prdx6-fused to TAT was purified to assess protective potential. Relative enzymatic activity was measured by NADPH/GSH/GSH reductase coupled-assay. Cultured LECs and rat/mouse lenses were pretreated with the Prdx6 protein (2 to 20μg/ml) and exposed to H2O2 or UVB radiation. Stability of transduced wt-and mut-Prdx6 in cells treated with Cycloheximide was examined by immunoblotting with HA antibody.
Results:
Aging cells had increased Sumo1 levels and Sumoylation of most proteins. Higher oxidative load aberrantly Sumoylated Prdx6 and Sp1, leading to loss of cellular function and stability. Prdx6K122/142R was more stable with enhance protective activity, Prdx6-deficient cells co-overexpressing Sumo1 and wt-Prdx6 were more prone to apoptosis than cells with Sumo1 plus Prdx6K122/142R. Delivery of TAT-linked-Prdx6K122/142R transduced to LECs and lenses, dramatically enhancing protection against oxidative stress and delaying lens opacity.
Conclusions:
Aberrant Sumoylation signaling is involved in dysregulation of Prdx6 during oxidative stress and aging. The process can be blocked by Prdx6K122/142R. This may offer a new approach for manipulating proteins to potentiate their activity against diseases linked to oxidative stress and aging.
Keywords: 634 oxidation/oxidative or free radical damage •
657 protein modifications-post translational •
424 antioxidants