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Poly-ADP ribose polymerase activates nuclear proteasome to degrade oxidatively damaged histones
O Ullrich1, T Reinheckel, N Sitte
1Clinics of Physical Medicine and Rehabilitation, Humboldt University, Berlin, Germany.
Abstract:
The 20S proteasome has been shown to be largely responsible for the degradation of oxidatively modified proteins in the cytoplasm. Nuclear proteins are also subject to oxidation, and the nucleus of mammalian cells contains proteasome. In human beings, tumor cells frequently are subjected to oxidation as a consequence of antitumor chemotherapy, and K562 human myelogenous leukemia cells have a higher nuclear proteasome activity than do nonmalignant cells. Adaptation to oxidative stress appears to be one element in the development of long-term resistance to many chemotherapeutic drugs and the mechanisms of inducible tumor resistance to oxidation are of obvious importance. After hydrogen peroxide treatment of K562 cells, degradation of the model proteasome peptide substrate suc-LLVY-MCA and degradation of oxidized histones in nuclei increases significantly within minutes. Both increased proteolytic susceptibility of the histone substrates (caused by modification by oxidation) and activation of the proteasome enzyme complex occur independently during oxidative stress. This rapid up-regulation of 20S proteasome activity is accompanied by, and depends on, poly-ADP ribosylation of the proteasome, as shown by inhibitor experiments, 14C-ADP ribose incorporation assays, immunoblotting, in vitro reconstitution experiments, and immunoprecipitation of (activated) proteasome with anti-poly-ADP ribose polymerase antibodies. The poly-ADP ribosylation-mediated activated nuclear 20S proteasome is able to remove oxidatively damaged histones more efficiently and therefore is proposed as an oxidant-stimulatable defense or repair system of the nucleus in K562 leukemia cells.
Insights
The nuclear 20S proteasome rapidly activates during oxidative stress in leukemia cells. This activation, mediated by poly-ADP ribosylation, enhances the removal of damaged histones, aiding tumor resistance.
Area of Science:
- Cellular Biology
- Biochemistry
- Molecular Oncology
Background:
- Oxidative stress damages nuclear proteins, impacting cell function and survival.
- Tumor cells, particularly during chemotherapy, experience significant oxidation.
- K562 leukemia cells exhibit higher nuclear proteasome activity, suggesting a role in oxidative stress response.
Purpose of the Study:
- Investigate the role and regulation of the nuclear 20S proteasome in response to oxidative stress in K562 leukemia cells.
- Determine the mechanism by which nuclear proteasome activity is enhanced under oxidative conditions.
- Elucidate the potential role of this activated proteasome in cellular defense against oxidative damage.
Main Methods:
- Hydrogen peroxide treatment of K562 cells.
- Assay of proteasome activity using peptide substrates (suc-LLVY-MCA) and oxidized histones.
- Analysis of poly-ADP ribosylation of the proteasome via inhibitor experiments, radiolabeling, immunoblotting, and in vitro reconstitution.
- Immunoprecipitation using anti-poly-ADP ribose polymerase antibodies.
Main Results:
- Oxidative stress significantly increased the degradation of oxidized histones and peptide substrates within minutes.
- Both substrate susceptibility and proteasome activation occurred independently during oxidative stress.
- Proteasome activation was dependent on poly-ADP ribosylation, confirmed by various biochemical assays and reconstitution experiments.
Conclusions:
- The nuclear 20S proteasome is rapidly activated by poly-ADP ribosylation in response to oxidative stress in K562 leukemia cells.
- This activated proteasome efficiently degrades oxidatively damaged histones.
- The poly-ADP ribosylation-mediated nuclear proteasome functions as an oxidant-stimulatable defense system in leukemia cells.
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