Influence of DNA binding on the degradation of oxidized histones by the 20S proteasome.
The 20S proteasome is localized in the cytosol and nuclei of mammalian cells. Previous work has shown that the cytosolic 20S proteasome is largely responsible for the selective recognition and degradation of oxidatively damaged cytosolic proteins. Since nuclear proteins are also susceptible to oxidative damage (e.g., from metabolic free radical production, ionizing radiation, xenobiotics, chemotherapy) we investigated the degradation of oxidatively damaged histones, in the presence and in the absence of DNA, by the 20S proteasome. We find that both soluble histones and DNA-bound histones are susceptible to selective proteolytic degradation by the 20S proteasome following mild oxidative damage. In contrast, more severe oxidative damage actually decreases the proteolytic susceptibility of histones. Soluble H1 showed the highest basal and maximal absolute proteolytic rates. Histone fraction H4 exhibited the greatest relative increase in proteolytic susceptibility following oxidation, almost 14-fold, and this occurred at a peroxide exposure of 5 mM. At the other end of the spectrum, histone H2A exhibited a maximal proteolytic response to H2O2 of only 6-fold, and this required an H2O2 exposure of 15 mM. An oxidation of reconstituted linear DNA plasmid-histone complex makes up to 95% of the histones bound to DNA susceptible to degradation, whereas undamaged protein-DNA complexes are not substrates for the proteasome. Severe oxidation by high concentrations of H2O2 appears to decreases the proteolytic susceptibility of histones due to the formation of cross-linked histone-DNA aggregates which appear to inhibit the proteasome. We conclude that the degradation of nuclear proteins is highly selective and requires prior damage of the substrate protein, such as that caused by oxidation.
Ullrich
O
O
Clinics of Physical Medicine and Rehabilitation, University Hospital Charité, Humboldt-University, Berlin, Germany.
Sitte
N
N
Sommerburg
O
O
Sandig
V
V
Davies
K J
KJ
Grune
T
T
D003546:Cysteine Endopeptidases / Q000378:metabolism*; D004247:DNA / Q000378:metabolism*; D004268:DNA-Binding Proteins / Q000378:metabolism; D004305:Dose-Response Relationship, Drug; D004587:Electrophoresis, Agar Gel; D004912:Erythrocytes; D006657:Histones / Q000378:metabolism*; D006801:Humans; D006861:Hydrogen Peroxide / Q000494:pharmacology; D007700:Kinetics; D009097:Multienzyme Complexes / Q000378:metabolism*; D010084:Oxidation-Reduction; D018384:Oxidative Stress; D046988:Proteasome Endopeptidase Complex; D011485:Protein Binding / Q000187:drug effects