Structures & Redox Chemistry in Sulfinic Acid Reduction

结构

基本信息

批准号：
8623135
负责人：
W TODD LOWTHER
金额：
$ 34.78万
依托单位：
WAKE FOREST UNIVERSITY HEALTH SCIENCES
依托单位国家：
美国
项目类别：
财政年份：
2005
资助国家：
美国
起止时间：
2005-08-01 至 2017-02-28
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/8623135
关键词：
Active Sites Address Affect Aging Alzheimer&apos s Disease Antioxidants Apoptosis Binding Biological Markers C-terminal Cardiovascular Diseases Catalysis Cell Culture Techniques Cell Death Chemistry Chemosensitization Chimera organism Complex DNA DNA Sequence Rearrangement Data Development Disease Disease Resistance Drug Metabolic Detoxication Enzymes Event Exhibits Future Glutathione Heart Human Hydrogen Peroxide In Vitro Individual Ionizing radiation Kinetics Lead Lipids Liver Malignant Neoplasms Mass Spectrum Analysis Mediating Mediator of activation protein Mitochondria Molecular Myocardial Infarction Nucleotides Oxidation-Reduction Oxidative Stress PTPN1 gene Parkinson Disease Process Protein phosphatase Proteins Reaction Reaction Time Reactive Oxygen Species Recombinants Reducing Agents Relative (related person)Reperfusion Injury Resistance Second Messenger Systems Signal Transduction Site Structure Structure-Activity Relationship Sulfinic Acids Sulfur Time Tissues Transgenic Organisms Up-Regulation Variant age related base cancer therapy chemotherapy comparative cysteinylcysteine design gene therapy innovation insight mutant novel overexpression oxidation prevent public health relevance repair enzyme repaired research study second messenger sulfenamide time use treatment strategy

项目摘要

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).

描述（由申请人提供）：在包括过氧化氢（H2O2）的活性氧中排毒中，典型的2-Cys过氧蛋白（PRX）是关键的抗氧化剂酶。在较低的浓度下，H2O2也被认为是细胞信号的重要介体。在这种情况下，PRX与H2O2的高细胞浓度和反应性使它们非常适合调节依赖氧化还原的信号传导事件。但是，通过与Cys硫酸Cys硫酸（Cys-SO2-）的高氧化可以使人2-CYS PRX灭活，这是许多衰老相关疾病和癌症的标志。通过酶硫蛋白（SRX）对高氧化和这些PRX的修复的敏感性有所不同。线粒体PRXIII是对高氧化的最具耐药性。令人惊讶的是，当存在在不同的氧化态（CYS-SH，CYS-S-S-CYS和CYS-SO2-）中时，人类PRX的结构几乎没有细节，对于此类酶的高氧化和SRX介导的修复的动力学知之甚少。我们已经表明，SRX利用一种新型的核苷酸结合基序和硫化学来减少PRX分子，并能够通过SRX修复高氧化PRXII的临界动力学中间体。另一方面，PRXIII在该区域表现出独特的C末端序列，该区域有望根据人类SRX7PRXI复合物的晶体结构直接与SRX接触。因此，我们假设PRXIII不仅由于其C末端和主动位点差异而对高氧化具有更大的抵抗力，而且它将与SRX具有独特的相互作用，可能会影响维修过程。对四个人类2-Cys PRX（PRXI-IV）的初步研究已经证实了在细胞培养中获得的结果，并表明PRXIII确实对高氧化具有最抗性。此外，我们在不同的氧化态下为PRXI IV产生了初步晶体，并通过时间分辨的质谱法进行了对PRXII和PRXIII高氧化的比较动力学研究。这些分析首次表明了prxii中分子内Cys磺酰胺中间体的形成。有趣的是，PRXIII并未在相同的反应条件下形成该物种，从而确定了一种潜在的情况，该方案可能对PRXI，PRXII和PRXIIV中的高氧化敏感性以及对PRXIII中高氧化的耐药性。鉴于PRXIII和SRX的转基因表达可防止心肌梗塞期间氧化应激诱导的凋亡和组织损伤，因此了解PRXS催化，高氧化和SRX修复的PRXS催化和动力学基础的理解是使用PRXIII和/或/或/或/或/或/或/或/或/或/或/或或SRX的未来设计。该提案的具体目的是研究人2-Cys PRX中高氧化的结构和动力学决定因素（AIM I），并研究SRX人类2-CYS PRX的修复机制（AIM2）。

项目成果

期刊论文数量（9）

专著数量（0）

科研奖励数量（0）

会议论文数量（0）

专利数量（0）

Unique Cellular and Biochemical Features of Human Mitochondrial Peroxiredoxin 3 Establish the Molecular Basis for Its Specific Reaction with Thiostrepton.

DOI：
10.3390/antiox10020150
发表时间：
2021-01-20
期刊：
Antioxidants (Basel, Switzerland)
影响因子：
0
作者：
Nelson KJ;Messier T;Milczarek S;Saaman A;Beuschel S;Gandhi U;Heintz N;Smalley TL;Lowther WT;Cunniff B
通讯作者：
Cunniff B

Mitochondrial peroxiredoxin 3 is more resilient to hyperoxidation than cytoplasmic peroxiredoxins.