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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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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.
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.
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.
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.
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