Role of antioxidant-1 in extracellular superoxide dismutase function and expression
Role of antioxidant-1 in extracellular superoxide dismutase function and expression
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DOI：
10.1161/01.res.0000162001.57896.66
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发表时间：
2005-04-15
影响因子：
20.1
通讯作者：
Fukai, T
中科院分区：
文献类型：
--
作者：
Jeney, V;Itoh, S;Fukai, T
细胞外超氧化物歧化酶（ecSOD或SOD3）是一种含铜酶，在脉管系统中高度表达。含铜酶的活性需要铜伴侣蛋白，然而将铜运输给SOD3的伴侣蛋白此前尚未确定。Atox1是一种被认为可将铜运输到反式高尔基体网络的铜伴侣蛋白。由于SOD3是通过反式高尔基体网络分泌的，我们试图确定Atox1是否作为SOD3的铜伴侣蛋白发挥作用。利用重组人SOD3，我们发现SOD3的比活性与其铜含量直接相关（R² = 0.99）。来自培养的Atox1（-/-）成纤维细胞的条件培养基中SOD3的比活性显著降低，但通过添加铜可恢复到野生型细胞的水平。这些结果表明，Atox1是将铜运输给SOD3以使其具有完全活性所必需的。出乎意料的是，在培养的Atox1（-/-）成纤维细胞中，SOD3的蛋白质和mRNA水平显著降低。这与SOD3转录速率的显著降低有关，但SOD3 mRNA的稳定性没有变化。Atox1的过表达在Atox1（-/-）和Atox1（+/+）细胞中均显著增加了SOD3 mRNA。这些发现表明Atox1正向调节SOD3转录。由于SOD3蛋白在动脉粥样硬化血管中上调，我们检测了ApoE（-/-）小鼠血管中Atox1的表达。对ApoE（-/-）小鼠进行的蛋白质印迹和免疫组织化学分析显示，在动脉粥样硬化内膜病变中，Atox1和SOD3的蛋白质水平均显著升高。总之，Atox1不仅作为SOD3的铜伴侣蛋白发挥作用，还作为SOD3转录的正向调节剂，可能在调节心血管系统的氧化应激中具有重要作用。
The extracellular superoxide dismutase (ecSOD or SOD3) is a copper-containing enzyme which is highly expressed in the vasculature. Copper-containing enzymes require copper chaperones for their activity however the chaperone which delivers copper to SOD3 has not previously been defined. Atox1 is a copper chaperone proposed to deliver copper to the trans-Golgi network. Because SOD3 is secreted via the trans-Golgi network, we sought to determine whether Atox1 acts as a copper chaperone for SOD3. Using recombinant human SOD3, we found that the specific activity of SOD3 directly correlates with its copper content (R-2 = 0.99). SOD3 specific activity in the conditioned medium from cultured Atox1(-/-) fibroblasts was markedly decreased, but could be recovered to that of wild-type cells by copper addition. These results indicated that Atox1 is required for delivering copper to SOD3 for its full activity. Unexpectedly, the protein and mRNA levels of SOD3 were dramatically decreased in cultured Atox1(-/-) fibroblasts. This was associated with a marked decrease in SOD3 transcription rate but no change in SOD3 mRNA stability. Overexpression of Atox1 markedly increased SOD3 mRNA in both Atox1(-/-) and Atox1(-/-) cells. These findings indicate that Atox1 positively regulates SOD3 transcription. Because SOD3 protein is upregulated in atherosclerotic vessels, we examined expression of Atox1 in vessels from ApoE(-/-) mice. Western and immunohistochemical analysis in ApoE(-/-) mice revealed that both Atox1 and SOD3 protein levels are markedly increased in atherosclerotic intimal lesions. In summary, Atox1 functions not only as a copper chaperone for SOD3 but also as a positive regulator for SOD3 transcription and may have an important role in modulating oxidative stress in the cardiovascular system.