Structures & Redox Chemistry in Sulfinic Acid Reduction

描述（由申请方提供）：典型的2-Cys过氧化物酶（Prxs）是活性氧（包括过氧化氢（H2 O2））解毒的关键抗氧化酶。在较低浓度下，H2 O2也被认为是细胞信号传导的重要介质。在这种情况下，Prxs与H2 O2的高细胞浓度和反应性使其非常适合调节氧化还原依赖性信号传导事件。然而，人2-Cys Prx可以通过过度氧化成Cys亚磺酸（Cys-SO2-）而失活，这是许多衰老相关疾病和癌症的标志。对过氧化的敏感性和由硫氧还蛋白（Srx）酶对这些Prx的修复是不同的。线粒体PrxIII对过氧化最有抵抗力。令人惊讶的是，当以不同的氧化态（Cys-SH、Cys-S-S-Cys和Cys-SO 2-）存在时，几乎没有关于人Prx的结构的细节，并且关于这类酶的过氧化和Srx介导的修复的动力学甚至知之甚少。我们已经表明，Srx利用一种新的核苷酸结合基序和硫化学减少Prx分子，并能够确定关键的动力学中间体在修复过氧化PrxII Srx。另一方面，PrxIII在预期与Srx直接接触的区域中表现出独特的C-末端序列，这是基于我们的人Srx 7 PrxI复合物的晶体结构。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能抵抗过氧化，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2-Cys Prxs（PrxI-IV）的初步研究已经证实了在细胞培养中获得的结果，并且已经表明PrxIII确实是最抗过氧化的。此外，我们已经产生了初步的晶体PrxI-IV在不同的氧化态，并进行比较动力学研究PrxII和PrxIII过氧化时间分辨质谱。这些分析首次显示了PrxII中分子内Cys次磺酰胺中间体的形成。有趣的是，PrxIII没有形成这种物质在相同的反应条件下，确定一个潜在的情况下，可能会赋予敏感性的PrxI，PrxII和PrxIV和抗过氧化的PrxIII。考虑到转基因表达的PrxIII和Srx的结果在心肌梗死期间保护免受氧化应激诱导的细胞凋亡和组织损伤，理解Prxs催化、过氧化和Srx修复的结构和动力学基础对于未来设计使用PrxIII和/或Srx变体进行基因治疗的新型治疗策略将是非常宝贵的。该提案的具体目的是研究人2-Cys Prxs（Aim I）中过氧化的结构和动力学决定因素，并研究Srx（Aim 2）对人2-Cys Prxs的修复机制。

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).