Sumoylation Motif Disruption Of Prdx6 (K122/142R) Enhances Protection Against Oxidative Stress In Lens Epithelial Cells By Potentiating GSH Peroxidase And aiPLA₂ Activities

Abstract

Purpose : Oxidative stress-evoked aberrant Sumoylation signaling causes loss of Peroxiredoxin (Prdx)6 expression and cell death. In this study we showed that disruption of Prdx6 Sumoylation sites K (Lysine)122/142R (Arginine) enhanced Prdx6’s protective efficacy by promoting its GSH peroxidase and acidic Ca²⁺ -independent phospholipase A₂ (aiPLA₂) activities in lens epithelial cells (LECs).

Methods : Prdx6 cDNA and its mutant (Mut) K122/142R cloned into pGFP or pTAT-HA vector were used to disrupt Prdx6’s redox-active site [cysteine (C)47 to isoleucine (I)], PLA₂ active sites (Ser32, His26 and Asp140 to Ala) and phosphorylation site, Thr177 to Ala by site directed mutagenesis. Wild type (Wt) and MutPrdx6-fused to TAT were purified to transduce into LECs overexpressing pGFP-Sumo1 and assessed protective potential against H₂O₂-driven stress. MTS and H2DCF-DA dye assays measured cell viability and ROS levels, respectively. TAT-Prdx6 or Prdx6^K122/142R or its Muts at C47I and/or PLA₂, and Prdx6^-/- LECs as well as human (h) LECs transfected/cotransfected with GFP-Prdx6 or GFP-Prdx6^K122/142R or its mutants at C47I and /or PLA₂ sites were used to determine relative enzymatic activities of GSH peroxidase and PLA₂ by commercially available kits. qPCR and Western analysis monitored expression of Prdx6 and Sumo1 in LECs.

Results : TAT-linked Prdx6^K122/142R transduced to Prdx6^-/- LECs and hLECs overexpressing Sumo1 or these cells transfected with pGFP-Prdx6^K122/142R were more stable and efficacious at cytoprotection than pGFP or TAT-linked WtPrdx6. Transduction or transfection of Prdx6^-/- LECs with Wt and MutC47I or Mut of the aiPLA₂ did not rescue cells. Prdx6^K122/142R showed GSH peroxidase enhanced by 40% and aiPLA₂ activity by 30%, demonstrating that Sumoylation reduced Prdx6’s protective efficacy. The PLA₂ activity of Prdx6 was dramatically decreased by 8 times in cells transfected with Thr177A mutant, suggesting Prdx6 phosphorylation is required for enhanced PLA₂ activity.

Conclusions : While aberrant Sumoylation of Prdx6 abates the ability of Prdx6 to protect LECs against oxidative stress, that ability can be improved by mutation at Sumo sites K122/142R. Application of Prdx6^K122/142R in the form of a TAT-fusion protein may open a promising and easily applicable intervention for oxidative stress-induced pathobiology and cataractogenesis.

This is an abstract that was submitted for the 2016 ARVO Annual Meeting, held in Seattle, Wash., May 1-5, 2016.