May 2005
Volume 46, Issue 13
Free
ARVO Annual Meeting Abstract  |   May 2005
TAT–Mediated Protein Transduction of Prdx6 into Lens Epithelial Cells and Ocular Lenses: A Tool to Postpone Cataractogenesis
Author Affiliations & Notes
  • D.P. Singh
    Ophthalmology, Univ of Nebraska Med Ctr, Omaha, NE
  • E. Kubo
    Ophthalmology, Univ of Fukui, Fukui, Japan
  • Y. Akagi
    Ophthalmology, Univ of Fukui, Fukui, Japan
  • N. Fatma
    Ophthalmology, Univ of Nebraska Med Ctr, Omaha, NE
  • Footnotes
    Commercial Relationships  D.P. Singh, None; E. Kubo, None; Y. Akagi, None; N. Fatma, None.
  • Footnotes
    Support  NIH Grant EY13394
Investigative Ophthalmology & Visual Science May 2005, Vol.46, 3858. doi:
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      D.P. Singh, E. Kubo, Y. Akagi, N. Fatma; TAT–Mediated Protein Transduction of Prdx6 into Lens Epithelial Cells and Ocular Lenses: A Tool to Postpone Cataractogenesis . Invest. Ophthalmol. Vis. Sci. 2005;46(13):3858.

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

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Abstract

Abstract: : Purpose: Reactive oxygen species (ROS) are considered a major mediator in the development of many age–related diseases including cataractogenesis. Previously, we have demonstrated that peroxiredoxin 6 (Prdx6) provides cellular protection and plays a role in maintaining cellular homeostasis by regulating ROS levels. In the present study, we investigated the TAT protein transduction domain–mediated transduction of Prdx6 into lens epithelial cells (LECs) and/or lenses in vitro and in vivo and ascertained its protective potential against oxidative stress. Methods: To evaluate the transduction ability and protective potential of Prdx6, we engineered Prdx6 linked to TAT protein transduction domain. A full length cDNA was cloned into Nco1and EcoR1 sites of pTAT–HA prokaryotic expression vector and recombinant protein was purified. mLECs and /or rat lenses were cultured with TAT–HA–Prdx6 protein of various concentration for variable period. Mutant Prdx6 was used as control. Oxidative stress was induced by addition of H2O2 and/or paraquat in cell culture. Subconjuctival injection was performed to deliver TAT–HA–Prdx6 into Shumiya cataract rat (SCR), to assess its ability in postponing cataractogenesis. Western blot and immunohistochemistry were used to confirm the internalization of Prdx6. MTS and trypan blue assays were done for cell viability. TUNEL and DAPI staining were performed to define cell death. H2DCFH–DA dye was used to monitor ROS levels. Results: Western and immunohistochemical analysis with anti–His antibody revealed the transduction of TAT–HA–Prdx6 into LECs/lenses within 30 min in a concentration dependent fashion, and biologically active. Recombinant protein was present in cells within 4 h following subconjuctival injection. Cell viability assay showed protection of LECs against paraquat (5–10mM) and H2O2 induced cyto–toxicity. Lenses cultured in presence of TAT–HA–Prdx6 did not show opacity compared to the control lenses following H2O2 exposure (100 or 200 µM). Sub–conjunctival administration of TAT–HA–Prdx6 (20µg/10µl) could postpone the progression of cataract in SCR rats. Conclusions: Systemic or local application of TAT–Prdx6 prolongs cellular survival and prevents the progression of cataract. Therefore, application of Prdx6 in the form of a TAT fusion protein may open a promising and easily applicable new tool for providing cellular protection against oxidative stress and to postpone cataractogenesis.

Keywords: cataract • apoptosis/cell death • antioxidants 
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