Figure 3.

Proteasome-dependent selective degradation of oxidized histones in nuclear extracts and the role of poly-ADP ribosylation. (A) The degradation of undamaged and H₂O₂-modified histones and suc-LLVY-MCA by lysates of H₂O₂-treated isolated nuclei from K562 hematopoietic cells. Nuclei were isolated, exposed to 1 mM H₂O₂ for times from 0 to 15 min (or sham exposed), lysed, and used to measure the proteolytic susceptibility of both untreated and H₂O₂-treated [³H]-labeled histones (added to the nuclear lysates) exactly as described in Materials and Methods. Isolated [³H]-labeled histones were oxidatively modified (or used as unmodified “native” histones) as described in Materials and Methods. The time scale represents the length of nuclei exposure (or sham-exposure) to H₂O₂ and the data points represent the means of three independent experiments (for which SD were always less than 10%). (Inset) The increase in the suc-LLVY-MCA degrading activity of nuclear lysates after treatment of nuclei with 1 mM H₂O₂ (for details see Fig. 2). (B) The degradation of H₂O₂-modified histones by K562 cell nuclear lysates is stimulated by pretreatment of nuclei with H₂O₂ but inhibited by both lactacystin and 3-ABA. Nuclei were isolated, exposed to 1 mM H₂O₂ for 15 min (or sham exposed), lysed, and used to measure the proteolytic susceptibility of both untreated and H₂O₂-treated [³H]-labeled histones, as described in Materials and Methods, in the presence or absence of the inhibitors 5 μM lactacystin (LC) or 1 mM 3-ABA. Where used, oxidized histones were treated with 15 mM hydrogen peroxide. All values represent the means ± SD of three independent experiments.