July 2018
Volume 59, Issue 9
Open Access
ARVO Annual Meeting Abstract  |   July 2018
Ginkgolic Acid Inhibits Aberrant Sumoylation-Mediated Dysregulation of Prdx6 and Sp1 to Protect Lens Epithelial Cells against Oxidative Injury
Author Affiliations & Notes
  • Dhirendra P Singh
    Ophthalmology and Visual Sciences, Univ of Neb Med Center, Omaha, Nebraska, United States
  • Bhavana Chhunchha
    Ophthalmology and Visual Sciences, Univ of Neb Med Center, Omaha, Nebraska, United States
  • gunho won
    Ophthalmology and Visual Sciences, Univ of Neb Med Center, Omaha, Nebraska, United States
  • Eri Kubo
    Ophthalmology, Kanazawa Medical University, Kanazawa, Ishikawa, Japan
  • Footnotes
    Commercial Relationships   Dhirendra Singh, None; Bhavana Chhunchha, None; gunho won, None; Eri Kubo, None
  • Footnotes
    Support  NEI, EY024589
Investigative Ophthalmology & Visual Science July 2018, Vol.59, 5652. doi:https://doi.org/
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      Dhirendra P Singh, Bhavana Chhunchha, gunho won, Eri Kubo; Ginkgolic Acid Inhibits Aberrant Sumoylation-Mediated Dysregulation of Prdx6 and Sp1 to Protect Lens Epithelial Cells against Oxidative Injury. Invest. Ophthalmol. Vis. Sci. 2018;59(9):5652. doi: https://doi.org/.

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      © ARVO (1962-2015); The Authors (2016-present)

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Abstract

Purpose : Dysregulation of proteins due to oxidative-induced aberrant Sumoylation, a post-translational modification process with small ubiquitin-like modifiers has been implicated for the development of several diseases. Previously we reported that, in response to oxidative stress, aberrant Sumoylation of antioxidant Peroxiredoxin 6 (Prdx6) and its transactivator specificity protein1 (Sp1) leads to their dysfunction and cell death. The current study tests if Sumoylation-inhibitor ginkgolic acid (GA) reverses aberrant Sumoylation-mediated dysregulation of Prdx6 and Sp1, thus preventing oxidative injury.

Methods : To examine the expression and Sumoylation status of Prdx6 and Sp1, Prdx6-/- and Prdx6+/+ mouse and human lens epithelial cells (LECs) were treated with 0–120μM GA, exposed to 0–500μM H2O2 at varying times, and assessed using qPCR, immunoblot, and Sandwich-Sumo1/Prdx6/Sp1 ELISA. ChIP and transactivation assays with Prdx6 promoters containing Sp1 sites tested effects of Sumoylation on DNA binding and the transactivation potential of Sp1. Relative protein stabilities were examined using GA-treated LECs exposed to H2O2 and cycloheximide. MTS and H2DCF-DA dye assays measured effects of GA on cell viability and ROS, respectively. To test the cellular fate and response of Sp1 and Prdx6, cells were exposed to 0–50μM of Betulinic acid (BA), a Sumoylation agonist. Statistical analysis was performed using two-tailed Student’s t-tests.

Results : In GA-treated LECs, ROS-dependent aberrant Sumoylation of Sp1 and Prdx6 significantly decreased (p<0.01), with increased levels of Sumoylated forms of both molecules, correlating well with a significant reduction in ROS and cell death compared to controls (p<0.01). GA treatment of redox-active Prdx6-/- cells reversed increased Sp1 Sumoylation. In GA-treated cells, transactivation activities of Sp1 significantly increased (~4-fold; p<0.001) with increased DNA binding. Conversely, BA-treated cells showed opposite results: increased Sumoylation-mediated-Sp1 and Prdx6 degradation. Thus, GA inhibited aberrant Sumoylation of ROS-induced Sp1 and Prdx6, thereby protecting LECs.

Conclusions : Our studies show not only the effects of aberrant protein Sumoylation on cellular fate during oxidative stress, but also offer a basis for creating targeted, small-molecule drugs to treat diseases from aberrant Sumoylation.

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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