Nitric oxide-induced changes in intracellular zinc homeostasis are mediated by metallothionein/thionein
Nitric oxide-induced changes in intracellular zinc homeostasis are mediated by metallothionein/thionein
复制标题
DOI：
10.1152/ajplung.00267.2001
复制
发表时间：
2002-02-01
影响因子：
4.9
通讯作者：
Pitt, BR
中科院分区：
文献类型：
--
作者：
St Croix, CM;Wasserloos, KJ;Pitt, BR
我们假设金属硫蛋白 (MT) 是一种对 Zn2+ 具有强亲和力的富含半胱氨酸的蛋白质，通过该蛋白质独特的硫醇盐簇封存或释放 Zn2+，在一氧化氮 (NO) 信号传导事件中发挥作用。将小鼠肺成纤维细胞 (MLF) 暴露于 NO 供体 S-亚硝基半胱氨酸，导致 Zn2+ 特异性荧光团 Zinquin 的荧光增加 20-30%，而 Zn2+ 螯合剂 N, N, N', N'-四-(2-吡啶甲基)乙二胺可迅速逆转这一现象。 MT 敲除导致 MLF 中不稳定 Zn2+ 不存在 NO 介导的增加，并且在通过腺病毒基因转移 MT 互补后恢复，这表明 MT 在 NO 调节 Zn2+ 稳态中发挥着关键作用。在绵羊肺动脉内皮细胞中获得的其他数据表明，MT 的脱辅基形式硫因 (T) 在完整细胞中作为 Zn2+ 结合蛋白发挥作用，因为 MT 的过度表达会抑制 NO 诱导的不稳定 Zn2+ 的变化，而补充 Zn2+ 可以逆转这种变化。此外，荧光共振能量转移数据表明，绿色荧光蛋白修饰的 MT 的过度表达可防止 NO 诱导的构象变化，这表明硫醇盐簇释放了 Zn2+。补充 Zn2+ 可以恢复这种效果。总的来说，这些数据表明 MT 介导 NO 诱导的细胞内 Zn2+ 变化，并表明 MT 与 T 的比例可以调节 Zn2+ 稳态以响应亚硝化应激。
We hypothesized that metallothionein (MT), a cysteine-rich protein with a strong affinity for Zn2+, plays a role in nitric oxide (NO) signaling events via sequestration or release of Zn2+ by the unique thiolate clusters of the protein. Exposing mouse lung fibroblasts (MLF) to the NO donor S-nitrosocysteine resulted in 20-30% increases in fluorescence of the Zn2+-specific fluorophore Zinquin that were rapidly reversed by the Zn2+ chelator N, N, N', N'-tetrakis-(2-pyridylmethyl) ethylenediamine. The absence of a NO-mediated increase in labile Zn2+ in MLF from MT knockouts and its restoration after MT complementation by adenoviral gene transfer inferred a critical role for MT in the regulation of Zn2+ homeostasis by NO. Additional data obtained in sheep pulmonary artery endothelial cells suggested a role for the apo form of MT, thionein (T), as a Zn2+-binding protein in intact cells, as overexpression of MT caused inhibition of NO-induced changes in labile Zn2+ that were reversed by Zn2+ supplementation. Furthermore, fluorescence-resonance energy-transfer data showed that overexpression of green fluorescent protein-modified MT prevented NO-induced conformational changes, which are indicative of Zn2+ release from thiolate clusters. This effect was restored by Zn2+ supplementation. Collectively, these data show that MT mediates NO-induced changes in intracellular Zn2+ and suggest that the ratio of MT to T can regulate Zn2+ homeostasis in response to nitrosative stress.