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Dhirendra P Singh, Bhavana Chhunchha, Eri Kubo; Prdx6 with Disrupted Sumoylation Sites, K122/142R Gains Function in Protecting Lens Epithelial Cells and Lenses by Abrogating Oxidative Stress-Induced Aberrant Sumoylation Signaling. Invest. Ophthalmol. Vis. Sci. 2014;55(13):3569.
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© ARVO (1962-2015); The Authors (2016-present)
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.
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.
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.
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.
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