May 2005
Volume 46, Issue 13
Free
ARVO Annual Meeting Abstract  |   May 2005
Identification of High Mobility Group Protein B1, a DNA–Binding Structural Chromosomal Protein, as a Novel Substrate of RPE Glutaredoxin
Author Affiliations & Notes
  • G. Hoppe
    Cole Eye Institute, Cleveland Clinic, Cleveland, OH
  • K.E. Talcott
    Cole Eye Institute, Cleveland Clinic, Cleveland, OH
  • S.K. Bhattacharya
    Cole Eye Institute, Cleveland Clinic, Cleveland, OH
  • J.W. Crabb
    Cole Eye Institute, Cleveland Clinic, Cleveland, OH
  • J.E. Sears
    Cole Eye Institute, Cleveland Clinic, Cleveland, OH
  • Footnotes
    Commercial Relationships  G. Hoppe, None; K.E. Talcott, None; S.K. Bhattacharya, None; J.W. Crabb, None; J.E. Sears, None.
  • Footnotes
    Support  NIH Grants EY06603, EY14239, EY15638, Fight for Sight, Foundation Fighting Blindness, CCF CIDA
Investigative Ophthalmology & Visual Science May 2005, Vol.46, 1594. doi:
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    • Get Citation

      G. Hoppe, K.E. Talcott, S.K. Bhattacharya, J.W. Crabb, J.E. Sears; Identification of High Mobility Group Protein B1, a DNA–Binding Structural Chromosomal Protein, as a Novel Substrate of RPE Glutaredoxin . Invest. Ophthalmol. Vis. Sci. 2005;46(13):1594.

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

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Abstract

Abstract: : Purpose: Protein sulfhydryls are reversibly modified by glutathione in response to oxidative stress, known as protein S–glutathionylation. Glutathione/disulfide exchange is catalyzed by the oxidoreductase glutaredoxin. To identify nuclear proteins that undergo redox–dependent S–glutathionylation and enzymatic de–glutathionylation in the retinal pigment epithelium (RPE), we pursued the identification of substrates of glutaredoxin. Methods: Selective oxidation of sulfhydryls was induced in cultured RPE cell lines (RPE–J or ARPE–19) by 5·10–4 M diamide. Nuclear proteins were isolated, treated with 2·10–6 M recombinant glutaredoxin, labeled with biotin–maleimide, purified, and identified by mass spectrometry. Sulfhydryl derivatization by mPEG–maleimide was used to determine the redox status of cysteine residues. Cellular localization and mobility of EGFP–fusion proteins were monitored using live fluorescent microscopy and fluorescence recovery after photobleaching (FRAP). Cysteine mutants were generated by PCR–based mutagenesis. Results: High mobility group protein B1 (Hmgb1), a DNA–binding structural chromosomal protein and transcriptional co–activator was identified as a substrate of glutaredoxin. Hmgb1 contains 3 cysteines, Cys23, 45, and 106 and their irreversible alkylation reduces Hmgb1 DNA binding. After oxidative stress, Cys23 and Cys45 readily form intramolecular disulfide bridge, whereas Cys106 remains in the reduced form. EGFP–tagged wild type Hmgb1 and endogenous Hmgb1 co–localized in the nucleus. Replacement of Cys23 and/or 45 with serines did not affect the nuclear distribution of the mutant proteins, while C106S and triple cysteine mutations impaired nuclear localization of Hmgb1. Intracellular mobility of Hmgb1 was severely impaired by the mutation of Cys106, but not of Cys23 or 45. Conclusions: Hmgb1 is a member of a large group of HMG–box containing transcription factors and co–activators, which include regulators of retinal development and survival. Hmgb1 and its homologue Hmgb2 are the only HMG–box proteins that contain cysteines which may be targets of reversible protein S–glutathionylation. Cys23 and 45 appear to confer redox–sensitivity to Hmgb1 by inducing conformational and functional changes in response to oxidative stress. Cys106 appears to govern the nucleocytoplasmic shuttling of Hmgb1.

Keywords: retinal pigment epithelium • oxidation/oxidative or free radical damage • protein structure/function 
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