September 2016
Volume 57, Issue 12
Open Access
ARVO Annual Meeting Abstract  |   September 2016
Sumoylation Motif Disruption Of Prdx6 (K122/142R) Enhances Protection Against Oxidative Stress In Lens Epithelial Cells By Potentiating GSH Peroxidase And aiPLA2 Activities
Author Affiliations & Notes
  • Dhirendra P Singh
    Ophthalmology and Visual Sciences, University of Nebraska Medical Center, Omaha, Nebraska, United States
  • Bhavana Chhunchha
    Ophthalmology and Visual Sciences, University of Nebraska Medical Center, Omaha, Nebraska, United States
  • Eri Kubo
    Ophthalmology, Kanazawa Medical University, Kanazawa, Ishikawa, Japan
  • Footnotes
    Commercial Relationships   Dhirendra Singh, None; Bhavana Chhunchha, None; Eri Kubo, None
  • Footnotes
    Support  NEI Grant EY024589
Investigative Ophthalmology & Visual Science September 2016, Vol.57, 2027. doi:
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      Dhirendra P Singh, Bhavana Chhunchha, Eri Kubo; Sumoylation Motif Disruption Of Prdx6 (K122/142R) Enhances Protection Against Oxidative Stress In Lens Epithelial Cells By Potentiating GSH Peroxidase And aiPLA2 Activities. Invest. Ophthalmol. Vis. Sci. 2016;57(12):2027.

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      © 2017 Association for Research in Vision and Ophthalmology.

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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 Prdx6K122/142R or its Muts at C47I and/or PLA2, and Prdx6-/- LECs as well as human (h) LECs transfected/cotransfected with GFP-Prdx6 or GFP-Prdx6K122/142R 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 Prdx6K122/142R transduced to Prdx6-/- LECs and hLECs overexpressing Sumo1 or these cells transfected with pGFP-Prdx6K122/142R 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. Prdx6K122/142R 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 Prdx6K122/142R 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.

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