Structures & Redox Chemistry in Sulfinic Acid Reduction

描述(由申请人提供)：典型的2-半胱氨酸过氧化还蛋白(Prxs)是包括过氧化氢(H_2O_2)在内的活性氧物种解毒的关键抗氧化酶。在较低浓度下，过氧化氢也被认为是细胞信号转导的重要介质。在这种背景下，Prxs的高细胞浓度和与过氧化氢的反应活性使它们非常适合于调节氧化还原依赖的信号事件。然而，人类的2-Cys Prxs可以通过过氧化生成Cys亚磺酸(Cys-SO2-)来灭活，Cys-SO2-是许多与衰老相关的疾病和癌症的标志。对过氧化的敏感性和硫氧还蛋白(SRX)酶对这些Prx的修复作用是不同的。线粒体PrxIII对过氧化的抵抗力最强。令人惊讶的是，关于人类Prx在不同氧化状态(Cys-SH、Cys-S-S-Cys和Cys-SO2-)下的结构细节很少，对这类酶的过氧化动力学和SRX介导的修复更是知之甚少。我们已经证明，SRX利用一个新的核苷酸结合基序和硫化学来还原Prx分子，并能够识别SRX修复高氧化PrxII的关键动力学中间体。另一方面，PrxIII在预计与SRX直接接触的区域显示了一个独特的C-末端序列，这是基于我们的人类Srx7PrxI复合体的晶体结构。因此，我们假设PrxIII不仅由于其C末端和活性位点的差异而更具抗氧化性，而且它将与SRX具有独特的相互作用，这可能影响修复过程。对四个人，2-半胱氨酸Prxs(PrxI-IV)的初步研究证实了在细胞培养中获得的结果，并表明PrxIII确实是最抗过氧化的。此外，我们还生成了PrxI-IV在不同氧化状态下的初步晶体，并用时间分辨质谱仪对PrxII和PrxIII的过氧化进行了比较动力学研究。这些分析首次表明在PrxII中形成了分子内半胱氨酸亚磺酰胺中间体。有趣的是，PrxIII没有在相同的反应条件下形成这种物种，从而确定了一种潜在的情景，该情景可能使PrxI、PrxII和PrxIV对过氧化敏感，而在PrxIII对过氧化具有抗性。鉴于PrxIII和SRX的转基因表达可防止心肌梗死时氧化应激诱导的细胞凋亡和组织损伤，了解Prxs催化、过氧化和SRX修复的结构和动力学基础将对未来利用PrxIII和/或SRX变体进行基因治疗的新治疗策略的设计具有重要价值。该方案的具体目的是研究人2-Cys Prxs(AIM2)过氧化的结构和动力学决定因素，并研究SRX(AIM2)修复人2-Cys Prxs的机制。

DESCRIPTION (provided by applicant): The typical 2-Cys peroxiredoxins (Prxs) are key antioxidant enzymes in the detoxification of reactive oxygen species including hydrogen peroxide (H2O2). At lower concentrations, H2O2 has also been recognized as an important mediator of cell signaling. In this context, the high cellular concentration and reactivity of Prxs with H2O2 makes them ideally suited to regulate redox-dependent signaling events. Human 2-Cys Prxs can be inactivated, however, through hyperoxidation to the Cys sulfinic acid (Cys-SO2-), a hallmark of many aging- related diseases and cancer. The sensitivity to hyperoxidation and the repair of these Prxs by the enzyme sulfiredoxin (Srx) differs. The mitochondrial PrxIII is the most resistant to hyperoxidation. Surprisingly, few details are available for the structures of the human Prxs when present in different oxidation states (Cys-SH, Cys-S-S-Cys, and Cys-SO2-), and even less is known about the kinetics of hyperoxidation and Srx-mediated repair for this class of enzymes. We have shown that Srx utilizes a novel nucleotide binding motif and sulfur chemistry to reduce the Prx molecule and were able to identify critical kinetic intermediates in the repair of hyperoxidized PrxII by Srx. PrxIII on the other hand exhibits a unique C-terminal sequence in the region that is expected to make direct contact with Srx, based on our crystal structure of the human Srx7PrxI complex. As such, we hypothesize that PrxIII is not only more resistant to hyperoxidation due to its C-terminus and active site differences, but also that it will have a unique interaction with Srx that may influence the repair process. Preliminary studies on the four human, 2-Cys Prxs (PrxI-IV) have confirmed the results obtained in cell culture and have shown that indeed PrxIII is the most resistant to hyperoxidation. In addition, we have generated preliminary crystals for PrxI-IV in different oxidation states and performed comparative kinetics studies for PrxII and PrxIII hyperoxidation by time-resolved mass spectrometry. These analyses have shown for the first time the formation of an intramolecular Cys sulfenamide intermediate in PrxII. Interestingly, PrxIII did not form this species under the same reaction conditions, identifying one potential scenario that may impart sensitivity to hyperoxidation in PrxI, PrxII, and PrxIV and resistance to hyperoxidation in PrxIII. Given that the transgenic expression of PrxIII and Srx results in protection against oxidative stress-induced apoptosis and tissue damage during myocardial infarction, an understanding of the structural and kinetics bases of Prxs catalysis, hyperoxidation, and repair by Srx will be invaluable for the future design of novel treatment strategies using PrxIII and/or Srx variants in gene therapy. The specific aims of the proposal are to investigate the structural and kinetic determinants of hyperoxidation in human 2-Cys Prxs (Aim I), and to investigate the repair mechanisms of human 2-Cys Prxs by Srx (Aim2).