Metal-catalyzed oxidation of the Werner syndrome protein causes loss of catalytic activities and impaired protein-protein interactions
Metal-catalyzed oxidation of the Werner syndrome protein causes loss of catalytic activities and impaired protein-protein interactions
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DOI：
10.1074/jbc.m706107200
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发表时间：
2007-12-14
影响因子：
4.8
通讯作者：
Bohr, Vilhelm A.
中科院分区：
文献类型：
--
作者：
Harrigan, Jeanine A.;Piotrowski, Jason;Bohr, Vilhelm A.
金属催化的氧化反应针对蛋白质金属结合口袋中的氨基酸。这种氧化反应通常会导致蛋白质的优先降解或随着年龄的增长而积累催化不活跃的蛋白质池。一直以来，氧化蛋白的水平都显示出随着年龄的增长而增加。节段性Werner综合征是由于Werner综合征蛋白(WRN)缺失所致。WRN是DNA解旋酶RecQ家族的一员，具有核酸外切酶和依赖于ATP的解旋酶活性。此外，WRN的每个解旋酶和核酸外切酶结构域都含有一个金属结合口袋。在这份报告中，我们研究了在铁或铜存在下金属催化的WRN的氧化。我们发现WRN在体外可以被铁氧化，但不被铜氧化。铁介导的氧化导致WRN解旋酶和核酸外切酶活性均受到抑制。WRN的氧化也抑制了与几个已知蛋白质伙伴的结合。此外，我们没有观察到20 S蛋白酶体在体外对氧化的WRN的降解。最后，细胞暴露在过氧化氢中会导致体内WRN的氧化。因此，我们的结果表明，在铁的存在下，WRN经历了金属催化的氧化，铁介导的WRN的氧化可能导致蛋白质的催化失活形式的积累，这可能有助于与年龄相关的表型。
Metal-catalyzed oxidation reactions target amino acids in the metal binding pocket of proteins. Such oxidation reactions generally result in either preferential degradation of the protein or accumulation of a catalytically inactive pool of protein with age. Consistently, levels of oxidized proteins have been shown to increase with age. The segmental, progeroid disorder Werner syndrome results from loss of the Werner syndrome protein (WRN). WRN is a member of the RecQ family of DNA helicases and possesses exonuclease and ATP-dependent helicase activities. Furthermore, each of the helicase and exonuclease domains of WRN contains a metal binding pocket. In this report we examined for metal-catalyzed oxidation of WRN in the presence of iron or copper. We found that WRN was oxidized in vitro by iron but not by copper. Iron-mediated oxidation resulted in the inhibition of both WRN helicase and exonuclease activities. Oxidation of WRN also inhibited binding to several known protein partners. In addition, we did not observe degradation of oxidized WRN by the 20 S proteasome in vitro. Finally, exposure of cells to hydrogen peroxide resulted in oxidation of WRN in vivo. Therefore, our results demonstrate that WRN undergoes metal-catalyzed oxidation in the presence of iron, and iron-mediated oxidation of WRN likely results in the accumulation of a catalytically inactive form of the protein, which may contribute to age-related phenotypes.