Stabilization Of Prdx6 Activity By Targeted Mutation Of Sumoylation Sites Protects Trabecular Meshwork Cells From Oxidative Damage

Abstract

Purpose : Abnormalities in trabecular meshwork (TM) caused by oxidative stress may lead to higher intraocular pressure and progression of glaucoma. We previously showed the involvement of aberrant Sumoylation of the antioxidant protein, peroxiredoxin (Prdx)6 in the pathobiology of TM cells. Here we test whether targeted mutation of Sumoylation sites in Prdx6 protects TM cells from oxidative damage.

Methods : TM cells isolated from one glaucomatous and seven normal donor eyes were exposed to H₂O₂ (0-200μM) for variable times. Subjects were in three age groups: young (3, 11 m), middle (39, 51, 54 y) and old (79, 88 y), plus glaucoma (56y).We assessed the effect of Prdx6 Sumoylation on ECM proteins, senescence markers p16/p21, by Sumo/Prdx6-ELISA, Western analysis, and qPCR, and examined correlation of these parameters with pathobiology of TM cell, if any. Prdx6 cDNA and mutant Prdx6 cDNA [at Sumo1 sites K (lysine) 122/142R (arginine) by site-directed mutagenesis] were cloned into the pTAT-vector. Purified TAT-Prdx6 protein was transduced into TM cells to assess its efficacy in abating pathobiology of cells overexpressing pSumo1. SA-βgal, MTS and H2DCF-DA dye assays measured cell senescence, viability and reactive oxygen species (ROS).The GSH-peroxidase and aiPLA2 activities were done by commercial kits. A two tailed Student’s t-test was used for statistical analysis.

Results : TM cells from the middle and old groups plus the glaucomatous subject showed reduced Prdx6 levels and increased Sumoylation of Prdx6 compared to younger subjects (p<0.001). The reduced Prdx6 activity was linked to increased ROS, SA-βgal activity, Sumo1 and ECM expression (p<0.05). The process was significantly increased (~3fold) in aged or redox-active cells due to loss of Prdx6 activity, mediated by aberrant Sumoylation (p<0.001). Cells transduced with Prdx6K122/142R were protected from oxidative damage having augmented GSH Peroxidase and aiPLA₂ activities. Moreover, TAT-Prdx6 K122/142R transduced TM cells, reversed ROS overshooting and abated TM cell injuries and senescence (p<0.001).

Conclusions : Findings reveal a mechanism regulated by Sumoylation of Prdx6 that rescued cells from oxidative stress-induced damage, suggesting a therapeutic potential for Sumoylation-deficient Prdx6 and offering a novel strategy for the treatment of glaucoma.

This is an abstract that was submitted for the 2017 ARVO Annual Meeting, held in Baltimore, MD, May 7-11, 2017.