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Bhavana Chhunchha, Eri Kubo, Dhirendra P Singh; Prdx6 Delivery Increases Lens Epithelial Cell Survival during Oxidative Stress by Reactivating Sp1-Prdx6 Transcription. Invest. Ophthalmol. Vis. Sci. 2018;59(9):3041.
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Increased oxidation and decreased antioxidant potential are major causes of age-related blindness. We have shown that aberrant Sumoylation of specificity protein1 (Sp1) by Sumo1 represses the antioxidant Peroxiredoxin 6 (Prdx6) during oxidative stress. Herein, we test if extrinsic delivery of Prdx6 linked to the TAT transduction domain restores Sp1 activity, repairs intrinsic Prdx6 transcription, and protects lens epithelial cells (LECs) from aging/oxidative stress.
The cDNA of Prdx6 and its mutant (site-directed mutagenesis at Sumo1 sites K122/142R) were cloned into pTAT-vector, purified, transduced into LECs isolated from Prdx6-/- mice (a model for aging) and normal donor lenses (n=12;16–68 years), and exposed to H2O2 (0–200μM) at variable times. ELISA, Western blot, and qPCR assessed the effect of TAT-Prdx6 delivery on endogenous Prdx6, the Sumoylation status of Sp1, and their relative abundance and activity. ChIP and transactivation with a Prdx6 promoter containing Sp1 sites were used to monitor the effect of TAT-Prdx6 and its mutant on the DNA binding and transactivation potential of Sp1. To assess the protective potential of Prdx6, MTS and H2DCF-DA dye assays measured cell viability and ROS levels, respectively. Statistical analysis was done using two-tailed Student’s t-tests.
Compared to younger subjects (<35years), the redox-active Prdx6-deficient mLECs and LECs of subjects >35 years showed reduced Sp1 and Prdx6 levels, increased Sumoylation of Sp1 and Prdx6 (p<0.05), and increased ROS (p<0.001), which jumped nearly three-fold in LECs exposed to oxidative stress (p<0.001). LECs transduced with Prdx6 or its mutant were protected from oxidative damage by removing excess ROS (p<0.001) and showed an increased abundance of intrinsic Sp1 and Prdx6. Intriguingly, TAT-Prdx6 or the transduced cells of its mutant restored Sp1-DNA interaction and showed a dramatic increase in Prdx6 transcription, suggesting Prdx6 delivery stabilizes Sp1 by removing ROS and ROS-mediated aberrant Sumoylation, thereby augmenting transactivation. Further, mutant/Sumoylation-deficient Prdx6 was more potent and stable than Prdx6.
These findings reveal dysregulation of Prdx6 due to aberrant Sumoylation of Sp1, leading to cell injury during aging/oxidative stress, which suggests the novel therapeutic potential of Sumoylation-deficient Prdx6 in oxidative-induced diseases.
This is an abstract that was submitted for the 2018 ARVO Annual Meeting, held in Honolulu, Hawaii, April 29 - May 3, 2018.
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