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首页> 外文期刊>Journal of Molecular Biology >S-glutathionylation of cysteine 99 in the APE1 protein impairs abasic endonuclease activity
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S-glutathionylation of cysteine 99 in the APE1 protein impairs abasic endonuclease activity

机译：APE1蛋白中半胱氨酸99的S-谷胱甘肽酰化会损害碱性内切核酸酶活性

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摘要

Human apurinic/apyrimidinic (AP) endonuclease 1 (APE1) is a central participant in the base excision repair pathway, exhibiting AP endonuclease activity that incises the DNA backbone 5′ to an abasic site. Besides its prominent role as a DNA repair enzyme, APE1 was separately identified as a protein called redox effector factor 1, which is able to enhance the DNA binding activity of several transcription factors through a thiol-exchange-based reduction-oxidation mechanism. In the present study, we found that human APE1 is S-glutathionylated under conditions of oxidative stress both in the presence of glutathione in vitro and in cells. S-glutathionylated APE1 displayed significantly reduced AP endonuclease activity on abasic-site-containing oligonucleotide substrates, a result stemming from impaired DNA binding capacity. The combination of site-directed mutagenesis, biochemical assays, and mass spectrometric analysis identified Cys99 in human APE1 as the critical residue for the S-glutathionylation that leads to reduced AP endonuclease activity. This modification is reversible by reducing agents, which restore APE1 incision function. Our studies describe a novel posttranslational modification of APE1 that regulates the DNA repair function of the protein.
机译：人嘌呤/ apyrimidinic（AP）核酸内切酶1（APE1）是碱基切除修复途径的主要参与者，表现出将DNA骨架5'切入无碱基位点的AP核酸内切酶活性。除了其作为DNA修复酶的突出作用外，APE1还被单独鉴定为一种称为氧化还原效应因子1的蛋白，该蛋白能够通过基于硫醇交换的还原氧化机制增强多种转录因子的DNA结合活性。在本研究中，我们发现人APE1在体外和细胞内都存在谷胱甘肽的情况下，在氧化应激条件下被S-谷胱甘肽化。 S-谷胱甘肽化的APE1在含无碱基位点的寡核苷酸底物上显示AP核酸内切酶活性显着降低，这是DNA结合能力受损的结果。定点诱变，生化分析和质谱分析相结合，将人APE1中的Cys99鉴定为S-谷胱甘肽化的关键残基，导致AP核酸内切酶活性降低。这种修饰可通过还原剂逆转，还原剂可恢复APE1切口功能。我们的研究描述了APE1的新型翻译后修饰，可调节蛋白质的DNA修复功能。

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