June 2013
Volume 54, Issue 15
Free
ARVO Annual Meeting Abstract  |   June 2013
Regulation of Sumoylation in Lens Epithelial Cells During Aging And Oxidative Stress: Sumoylation Reduces LEDGF’s Stability And DNA-Binding Efficiency
Author Affiliations & Notes
  • Bhavana Chhunchha
    Ophthalmology & Visual Sciences, Univ of Nebraska Medical Center, Omaha, NE
  • Nigar Fatma
    Ophthalmology & Visual Sciences, Univ of Nebraska Medical Center, Omaha, NE
  • Eri Kubo
    Ophthalmology, Kanazawa Medical University, Kanazawa, Japan
  • Dhirendra Singh
    Ophthalmology & Visual Sciences, Univ of Nebraska Medical Center, Omaha, NE
Investigative Ophthalmology & Visual Science June 2013, Vol.54, 2964. doi:
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      Bhavana Chhunchha, Nigar Fatma, Eri Kubo, Dhirendra Singh; Regulation of Sumoylation in Lens Epithelial Cells During Aging And Oxidative Stress: Sumoylation Reduces LEDGF’s Stability And DNA-Binding Efficiency. Invest. Ophthalmol. Vis. Sci. 2013;54(15):2964.

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

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Abstract

Purpose: Lens Epithelium-Derived Growth Factor (LEDGF) binds DNA/proteins and regulates survival signaling. It is a target for Small Ubiquitin-like Modifier (Sumo)1 modification. We investigated the regulation of global Sumoylation in human and mouse lenses /lens epithelial cells (LECs) during aging and oxidative stress, and explored the effect of Sumoylation on LEDGF’s expression/stability, DNA-binding activity and cellular response.

Methods: Lenses and LECs from aging human subjects (NDRI, PA) and aging mouse treated with H2O2 (0 to 200μM) were used to examine global Sumoylation, and Sumoylation of LEDGF by using immunoprecipitation and Sumoylation Assay (Epigentek), and expression of Hsp27/αB-crystallin, Sumo1 and Sumo-specific protease, Senp1 by real-time PCR, Western analysis. Prdx6-deficient LECs were used as a model for aging. Gelshift and CAT assays with Hsp27/αB-crystallin promoter monitored the effect of Sumoylation on LEDGF’s DNA-binding activities. Sumo1-LEDGF fusion protein was made by cloning LEDGF cDNA into eukaryotic, pSUMO1Star (LifeSensor) and prokaryotic, pSUMO vectors. Ledgf-/- cells transfected with pSUMOStar-LEDGF or its mutant treated or untreated with cycloheximide was used to assess the effect of Sumoylation on LEDGF stability and cell viability by Western blot and MTS assay, respectively.

Results: Sumoylation increased in most of the protein in aging LECs/lenses, consistent with increased Sumo1 expression and decreased Senp1 expression and activity. Sumoylation patterns of proteins in Prdx6-/- cells were similar to those in aging lenses/cells. Mild oxidative stress led to reduce most Sumo1 conjugates, while higher oxidative stress enhanced Sumo1-conjugates. Sumoylation of LEDGF reduced its stability and decreased cellular survival and Hsps expression as evidenced by ectopic expression of Sumo1-LEDGF in Ledgf-/- cells. Downregulation of Hsp27/αB-crystallin and reduced cell survival was due to inhibition of DNA-binding activity of LEDGF Sumoylation.

Conclusions: Aging and oxidative stress regulate Sumoylation -deSumoylation, and involve Sumoylation of LEDGF that lead to suppression of its survival activity, by impairing its DNA-binding efficiency and stability. LEDGF Sumoylation may become a target for therapeutic intervention against cataractogenesis or disorders associated with oxidative stress and age.

Keywords: 634 oxidation/oxidative or free radical damage • 739 transcription factors • 449 cell survival  
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