Comparative resistance of the 20S and 26S proteasome to oxidative stress
Comparative resistance of the 20S and 26S proteasome to oxidative stress
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DOI：
10.1042/bj3350637
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发表时间：
1998-11-01
影响因子：
4.1
通讯作者：
Grune, T
中科院分区：
文献类型：
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作者：
Reinheckel, T;Sitte, N;Grune, T
氧化修饰的铁蛋白被20 S蛋白酶体选择性识别和降解。浓度高于10 μ cmol/mg蛋白质的过氧化氢（H2 O2）能够防止蛋白水解降解。将蛋白酶暴露于大量氧化剂（H2 O2、过氧亚硝酸盐和次氯酸盐）抑制20 S蛋白酶体对荧光肽琥珀酰-亮氨酸-亮氨酸-缬氨酸-酪氨酸-甲基香豆酰胺（Suc-LLVY-MCA）的酶活性，以及正常和氧化剂处理的铁蛋白的蛋白水解降解。使用40 μ mol H2 O2/mg蛋白酶体实现了对蛋白质底物降解的50%抑制。电泳分析显示，在120 μ mol的H2 O2/mg的蛋白酶体的浓度的酶的组成没有变化。在进一步的实验中，人们发现26 S蛋白酶体（蛋白酶体系统的ATP和遍在蛋白依赖性形式）更容易受到氧化应激的影响。然而，12 μ mol H2 O2/mg对20 S蛋白酶体降解荧光肽Suc-LLVY-MCA的抑制率为50%，3 μ mol H2 O2/mg足以抑制ATP刺激的26 S蛋白酶体降解50%。这种活性损失可能伴随着非变性凝胶中条带强度的损失。因此，我们得出结论，20 S蛋白酶体比ATP和泛素依赖的26 S蛋白酶体更能抵抗氧化应激。此外，我们研究了H2 O2处理后K562细胞中这两种蛋白酶的活性。来自K562细胞的裂解物能够以ATP非依赖性方式以比非氧化铁蛋白更高的速率降解氧化铁蛋白。这种效果可以遵循，即使在用H2 O2处理的细胞浓度高达2 mM。lactacystin敏感的ATP刺激的降解的荧光肽Suc-LLVY-MCA下降，用1 mM H2 O2处理的细胞后，获得相同的水平，没有ATP刺激。因此，我们得出结论，20 S蛋白酶体的各种监管机构的监管发生在氧化应激。这为20 S蛋白酶体在哺乳动物细胞的二级抗氧化防御中的作用提供了进一步的证据。
Oxidatively modified ferritin is selectively recognized and degraded by the 20S proteasome. Concentrations of hydrogen peroxide (H2O2) higher than 10 mu cmol/mg of protein are able to prevent proteolytic degradation. Exposure of the protease to high amounts of oxidants (H2O2, peroxynitrite and hypochlorite) inhibits the enzymic activity of the 20S proteasome towards the fluorogenic peptide succinyl-leucine-leucine-valine-tyrosine-methylcoumarylamide (Suc-LLVY-MCA), as well as the proteolytic degradation of normal and oxidant-treated ferritin. Fifty per cent inhibition of the degradation of the protein substrates was achieved using 40 mu mol of H2O2/mg of proteasome. No change in the composition of the enzyme was revealed by electrophoretic analysis up to concentrations of 120 mu mol of H2O2/mg of proteasome. In further experiments, it was found that the 26S proteasome, the ATP- and ubiquitin-dependent form of the proteasomal system, is much more susceptible to oxidative stress. Whereas degradation of the fluorogenic peptide, Suc-LLVY-MCA, by the 20 S proteasome was inhibited by 50 % with 12 mu mol of H2O2/mg, 3 mu mol of H2O2/mg was enough to inhibit ATP-stimulated degradation by the 26S proteasome by 50 %. This loss in activity could be followed by the loss of band intensity in the non-denaturing gel. Therefore we concluded that the 20S proteasome was more resistant to oxidative stress than the ATP- and ubiquitin-dependent 26S proteasome. Furthermore, we investigated the activity of both proteases in K562 cells after H2O2 treatment. Lysates from K562 cells are able to degrade oxidized ferritin at a higher rate than non-oxidized ferritin, in an ATP-independent manner. This effect could be followed even after treatment of the cells with H2O2 up to a concentration of 2 mM. The lactacystin-sensitive ATP-stimulated degradation of the fluorogenic peptide Suc-LLVY-MCA declined, after treatment of the cells with 1 mM H2O2, to the same level as that obtained without ATP stimulation. Therefore, we conclude that the regulation of the 20 S proteasome by various regulators takes place during oxidative stress. This provides further evidence for the role of the 20S proteasome in the secondary antioxidative defences of mammalian cells.