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G. Hoppe, Y. Chai, K. West, J.E. Sears; Glutathione as a Second Messenger of Oxidation: Hsc70 Is a Substrate of Glutaredoxin-1 . Invest. Ophthalmol. Vis. Sci. 2003;44(13):1629.
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© ARVO (1962-2015); The Authors (2016-present)
Purpose: To determine the protein S-glutathiolated substrates of glutaredoxin-1 (grx-1) in cultured human retinal pigment epithelial cells. Methods: Protein-S-glutathiolation was induced in ARPE-19 cells by treatment with 250 µM diamide following pre-loading of cells with 14 µM reduced recombinant grx-1. Reversible glutathiolation by grx-1 was detected using monoclonal antibody specific for glutathione adduct and heat shock cognate protein 70 (Hsc70) identified by Q-TOF mass spectrometry. A luciferase aggregation assay was used to test Hsc70 activity in various redox states as well as in the presence of grx-1. Results: A 70 kD band that demonstrated reversible glutathiolation by grx-1 was identified as Hsc70. Recombinant Hsc70 was glutathiolated in vitro by oxidized glutathione, and Hsc70 protein-S-glutathiolation (Hsc70-SSG) reversed by reduced grx-1. Hsc70-SSG was twice as effective in preventing luciferase aggregation at 42 °C than reduced Hsc70. Pre-incubation with grx-1 enhanced the activity of Hsc70-SSG. Conclusions: Glutathione may become adducted to Hsc70 in order to enhance the activity of this heat shock protein under oxidative stress. The synergistic effect of grx-1 and Hsc70-SSG suggests that glutathiolation acts as a signal for cooperative binding between these two proteins to enhance chaperone activity.
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