DJ-1 is known to play neuroprotective roles by eliminating reactive oxygen species (ROS) as an antioxidant protein. However, the molecular mechanism of DJ-1 function has not been well elucidated. This study explored the structural and functional changes of DJ-1 in response to oxidative stress. We found that Cys46 is also reactive cysteine residue in DJ-1, which was identified employing an NPSB-B chemical probe that selectively reacts with redox sensitive cysteine sulfhydryl. Peroxidatic Cys46 readily formed an intra-disulfide bond with resolving Cys53, which was identified with nanoUPLC-ESI-q-TOF tandem mass spectrometry (MS/MS) employing DBond algorithm under the non-reducing condition. We also found that Cys46-Cys53 disulfide crosslinking affects the oxidative state of the third Cys106, which shows the crosstalk among three cysteine residues of DJ-1. Furthermore, we demonstrated that DJ-1 C46A mutant, not forming Cys46-Cys53 intra-disulfide bond, lost structural stability of DJ-1 employing hydrogen/deuterium exchange-mass spectrometry (HDX-MS) analysis. All three Cys mutants lost antioxidant activities in SN4741 cell, a dopaminergic neuronal cell, unlike wild type DJ-1. These findings suggest that DJ-1 regulates its structure and activities by concerted oxidative modifications of three cysteine residues. These studies broaden the understanding of regulatory mechanisms of DJ-1 that operate under oxidative conditions.

中文翻译：

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逐步氧化在DJ-1的结构和功能调节中起关键作用

已知DJ-1通过消除作为抗氧化剂的活性氧（ROS）发挥神经保护作用。但是，尚未很好地阐明DJ-1功能的分子机制。这项研究探讨了DJ-1响应氧化应激的结构和功能变化。我们发现Cys46也是DJ-1中的反应性半胱氨酸残基，已使用可与氧化还原敏感的半胱氨酸巯基选择性反应的NPSB-B化学探针进行了鉴定。过氧化物Cys46容易与解析的Cys53形成一个二硫键，在非还原条件下使用DBond算法通过nanoUPLC-ESI-q-TOF串联质谱（MS / MS）进行了鉴定。我们还发现Cys46-Cys53二硫键交联会影响第三个Cys106的氧化态，该图显示了DJ-1的三个半胱氨酸残基之间的串扰。此外，我们证明了不形成Cys46-Cys53内二硫键的DJ-1 C46A突变体使用氢/氘交换质谱（HDX-MS）分析失去了DJ-1的结构稳定性。与野生型DJ-1不同，所有三个Cys突变体都在多巴胺能神经元SN4741细胞中失去了抗氧化活性。这些发现表明，DJ-1通过三个半胱氨酸残基的协同氧化修饰来调节其结构和活性。这些研究拓宽了对在氧化条件下运行的DJ-1调节机制的理解。与野生型DJ-1不同，所有三个Cys突变体都在多巴胺能神经元SN4741细胞中失去了抗氧化活性。这些发现表明，DJ-1通过三个半胱氨酸残基的协同氧化修饰来调节其结构和活性。这些研究拓宽了对在氧化条件下运行的DJ-1调节机制的理解。与野生型DJ-1不同，所有三个Cys突变体都在多巴胺能神经元SN4741细胞中失去了抗氧化活性。这些发现表明，DJ-1通过三个半胱氨酸残基的协同氧化修饰来调节其结构和活性。这些研究拓宽了对在氧化条件下运行的DJ-1调节机制的理解。