March 2012
Volume 53, Issue 14
Free
ARVO Annual Meeting Abstract  |   March 2012
Sumoylation Modulates Prdx6 Cytoprotective Activity and Inhibits Its Sp1-Dependent Transcription
Author Affiliations & Notes
  • Bhavana Chhunchha
    Ophthalmology and Visual Sciences, Univ of Neb Med Center, Omaha, Nebraska
  • Nigar Fatma
    Ophthalmology and Visual Sciences, Univ of Neb Med Center, Omaha, Nebraska
  • Eri Kubo
    Dept of Ophthalmology, Kanazawa Medical University, Kahoku-gun, Japan
  • Biju Bhargavan
    Ophthalmology and Visual Sciences, Univ of Neb Med Center, Omaha, Nebraska
  • Dhirendra P. Singh
    Ophthalmology and Visual Sciences, Univ of Neb Med Center, Omaha, Nebraska
  • Footnotes
    Commercial Relationships  Bhavana Chhunchha, None; Nigar Fatma, None; Eri Kubo, None; Biju Bhargavan, None; Dhirendra P. Singh, None
  • Footnotes
    Support  NIH Grant EY013394 and EY017613
Investigative Ophthalmology & Visual Science March 2012, Vol.53, 1071. doi:
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      Bhavana Chhunchha, Nigar Fatma, Eri Kubo, Biju Bhargavan, Dhirendra P. Singh; Sumoylation Modulates Prdx6 Cytoprotective Activity and Inhibits Its Sp1-Dependent Transcription. Invest. Ophthalmol. Vis. Sci. 2012;53(14):1071.

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

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Abstract

Purpose: : Cytoprotective protein Peroxiredoxin 6 (Prdx6) has been shown to postpone oxidative stress-induced disorders and delay cataract by optimizing reactive oxygen species (ROS) and gene expression. Using lens epithelial cells (LECs) from normal human or rat lenses facing oxidative stress or Prdx6-depleted LECs coupled with biochemical analyses, we identified a novel posttranslational modification of Prdx6 and its transactivator, Sp1, namely Sumoylation, and found that Sumo (Small Ubiquitin-like Modifier)-1 regulated Prdx6 activity and expression.

Methods: : Web-based analysis (Sumoplot) was used to spot out putative Sumo-1 motif. His-tagged-Prdx6, GFP-Prdx6, pCMV-Sp1and EGFP-Sumo-1 were prepared for in vitro and in vivo Sumoylation assay. To identify Sumo-1 motif, Site-Directed Mutagenesis was used to abolish Sumo-1 motifs, (lysine [K] to arginine [R]). Prdx6-/- LECs and LECs facing oxidative stress were used for cotransfection and immunoprecipitation assays with anti-Sumo-1 or Prdx6 or GFP or Sp1 antibody. Prdx6 promoter (-839/+109) and mutant constructs at all three Sp1 sites linked to CAT were prepared. Gel-shift and transactivation assays monitored effects of Sumo-1 on Sp1 regulation of Prdx6 transcription. Real-time PCR and Western analysis were done to monitor mRNA and protein expression of Prdx6, Sumo-1 and Sp1 in human lenses or LECs facing oxidative stress. MTS and TUNEL assays determined cell survival and apoptosis.

Results: : SUMOplot pointed out four putative Sumo-1 motifs, Prdx6-K122 and K142 in Prdx6 protein. Point mutation and in vitro and in vivo Sumoylation assays showed that K122 and K142 are major Sumo-1 conjugation sites in Prdx6. Cotransfection assay disclosed that Sumo-1 downregulated Prdx6 transcription, which was dependent on Sumoylation of Sp1, reduced Sp1 protein and thereby decreased DNA binding activity to its sites (-17/-27, -61/-69, and -78/-90) in Prdx6 promoter. LECs overexpressing Sumo-1 displayed reduced expression and activity of Prdx6 and Sp1 mRNA and protein, and were highly susceptible to oxidative stress-induced apoptosis. A decline in Prdx6 and Sp1 expression was observed in Prdx6-/- cells and in aging as well as cataractous lenses, and was correlated with increased expression of Sumo-1 and decreased DNA binding activity of Sp1.

Conclusions: : Our findings revealed involvement of Sumoylation in regulating Prdx6 activity and expression. This study may provide a foundation for a strategy to repair deleterious oxidative signaling generated by reduced activity of Prdx6 by controlling the dynamic process of Sumoylation.

Keywords: antioxidants • protein modifications-post translational • oxidation/oxidative or free radical damage 
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