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MR Hosler, ST Wang-Su, BJ Wagner; Effect of Hydrogen Peroxide on the Peptidase Activities and 2D Gel Pattern of the 20S Proteasome in Human Lens Epithelial Cells (HLE-B3) . Invest. Ophthalmol. Vis. Sci. 2002;43(13):2370.
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© ARVO (1962-2015); The Authors (2016-present)
Purpose: The proteasome enzyme is an important component of the cellular oxidative stress response. This study provides details of the effects of oxidative stress on the proteasome enzyme. The observed effect on the three proteasomal peptidase activities and on the proteasome 2D gel pattern help to further understand the proteasome’s potential role in cataract formation. Methods: Cytosolic extracts were prepared from HLE-B3 cells treated with H2O2. Activity was assayed in these extracts with three peptides specific for the three distinct proteasomal peptidase activities. Proteasomal cleavage of these three substrates causes release of the flourogenic molecule, aminomethylcoumarin (AMC). The liberated AMC was quantitated upon 400nm excitation and 440nm emission. Observation of 20S proteasome 2D gel patterns required 35S labelling of HLE-B3 cells followed by immunoprecipitation with the monoclonal antibody MCP21 (Hybridoma 11: 507, 92). The radiolabeled, immunoprecipitated enzyme was separated in the first dimension on an immobilized non-linear pH gradient from pH 3.0 to pH 10.0. The first dimension gels were directed opposed to SDS-polyacrylamide gels, and separated at 200 volts in the second dimension. Second dimension gels were dried on a standard 80ºC vacuum gel drier for autoradiography in a Phosphorimager plate. Plates were scanned on a Molecular dynamics phosphorimager using ImageQuant 5.0 software. Results: H2O2 treatment of HLE-B3 cells caused a differential decrease in the three proteasome activities. The most sensitive activity is the chymotrypsin-like activity, while the peptidylglutamyl peptide hydrolysing and trypsin-like activities are less sensitive. Similar treatment of the HLE-B3 cells with H2O2 caused no significant change in newly synthesized 20S proteasome as seen by simultaneous H2O2 treatment with 35S radiolabeling. Conclusion: The described pattern of H2O2-induced proteasome inhibition, in the absence of changes in newly synthesized proteasome, suggest that the H2O2 may directly modify this enzyme chemically. Such a modification may be a mechanism through which oxygen radicals contribute to the accumulation of damaged proteins that may lead to cataract in the human lens.
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