Structures & Redox Chemistry in Sulfinic Acid Reduction

描述（由申请人提供）：典型的2-Cys过氧化物还毒素（Prxs）是活性氧解毒的关键抗氧化酶，包括过氧化氢（H2O2）。在较低浓度下，H2O2也被认为是细胞信号传导的重要介质。在这种情况下，Prxs的高细胞浓度和与H2O2的反应性使它们非常适合调节氧化还原依赖的信号事件。然而，人类2-Cys Prxs可以通过过度氧化生成Cys亚磺酸（Cys- so2 -）而失活，这是许多与衰老相关的疾病和癌症的标志。对高氧化的敏感性和这些Prxs被硫氧还蛋白（Srx）酶修复是不同的。线粒体PrxIII对高氧化的抵抗力最强。令人惊讶的是，很少有关于人类Prxs在不同氧化状态（Cys-SH， Cys-S-S-Cys和Cys-SO2-）下的结构的细节，甚至更少知道这类酶的高氧化动力学和srx介导的修复。我们已经证明Srx利用一种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，根据我们的人类Srx7PrxI复合物的晶体结构，PrxIII在该区域显示出独特的c端序列，预计与Srx直接接触。因此，我们假设PrxIII不仅由于其c端和活性位点的差异而具有更强的抗高氧化性，而且它与Srx具有独特的相互作用，可能会影响修复过程。对四种人类2-Cys Prxs （PrxI-IV）的初步研究证实了在细胞培养中得到的结果，并表明PrxIII确实是最抗高氧化的。此外，我们已经生成了不同氧化状态下PrxI-IV的初步晶体，并通过时间分辨质谱法对PrxII和PrxIII的高氧化进行了比较动力学研究。这些分析首次表明在PrxII中形成了分子内的Cys亚胺中间体。有趣的是，PrxIII并没有在相同的反应条件下形成这个物种，这表明PrxI、PrxII和PrxIV可能对高氧化敏感，而PrxIII对高氧化具有抗性。考虑到PrxIII和Srx的转基因表达可以防止心肌梗死期间氧化应激诱导的细胞凋亡和组织损伤，了解Prxs催化、高氧化和Srx修复的结构和动力学基础将对未来设计使用PrxIII和/或Srx变异体进行基因治疗的新治疗策略具有重要意义。该提案的具体目的是研究人类2-Cys Prxs高氧化的结构和动力学决定因素（Aim 1），并研究Srx对人类2-Cys Prxs的修复机制（Aim2）。

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).