Protein S-thiolations/trans-thiolations and oxidative stress

细胞氧化还原防御机制不能缓解的活性氧（ROS）的过度形成导致氧化应激。这种情况与许多重要的病理生物学状态有关，如细胞/器官毒性、衰老和主要疾病，如糖尿病和癌症。生物体已经开发出复杂的生化途径来控制ROS水平并减轻潜在的损害。这些包括肽/蛋白质硫醇氧化还原化学，抗氧化剂蛋白质和丰富的小内源性和外源性抗氧化剂氧化还原对。我们对这些低分子量氧化还原对如何调节蛋白质S-巯基化学的理解是有限的。深入了解它们与硫醇的详细反应对于理解作用机制、它们在硫醇氧化还原状态/氧化应激中的作用以及最终开发有效的治疗方法以对抗失调的ROS形成是重要的。三个基本的巯基相互作用的氧化还原对;维生素C（抗坏血酸）/脱氢抗坏血酸（DHA），谷胱甘肽（GSH）/谷胱甘肽二聚体（GSSG）和醌醇/醌，并将在本研究计划的主要重点调查。选择这些机构的原因如下：维生素C和GSH是哺乳动物系统中的主要抗氧化剂。醌类在所有毒性反应中占很大比例。后一种情况也更复杂，因为醌/醌氧化还原对与充分描述的抗氧化剂保护作用有关。因此，本研究计划的主要长期目标是阐明氧化还原可回收的小分子氧化剂（如醌醇/醌）影响蛋白质S-巯基化化学的机制。这包括蛋白质修饰/巯基交换反应以及这些修饰如何通过调节酶活性和毒性结果影响氧化还原稳态。为了阐明抗氧化剂/氧化剂对硫醇氧化还原化学的影响，将进行以下目标：1）通过阐明DHA-硫醇加合物的形成和在DHA水平升高的情况下鉴定细胞和血液中的S-硫醇化蛋白来详细表征DHA介导的S-硫醇化。2)与细胞氧化还原蛋白的GSH交换反应的表征和3）细胞中醌硫醇反应以及与作为这种反应的靶的氧化还原蛋白的醌硫醇反应的全面研究。这些研究将利用细胞实验和分析技术，如液相色谱法，质谱法，NMR，包括蛋白质组学方法。预期的结果将详细了解S-硫醇化和氧化还原法规的复杂性。这些结果将进一步加深我们对氧化还原对和蛋白质S-巯基化之间相互作用的认识。这些知识预计将是开发潜在标记物和调节氧化应激的新方法的第一个重要步骤。

Excessive formation of reactive oxygen species (ROS) that cannot be relieved by cell redox defence mechanisms leads to oxidative stress. This condition is associated with a multitude of significant pathobiological states such as cell/organ toxicity, ageing and major diseases such as diabetes and cancer. Organisms have developed complex biochemical pathways to control ROS levels and mitigate potential damage. These include peptide/protein thiol redox chemistry, antioxidant proteins and abundant small endo- and exogenous antioxidant redox couples. Our understanding about how these low molecular weight redox couples regulate protein S-thiol chemistry is limited. Insight into their detailed reactions with thiols is important in order to understand the mechanism(s) of action, their role in thiol redox status/oxidative stress and ultimately for the development of effective therapies to counteract deregulated ROS formation. Three essential thiol interacting redox couples are; vitamin C (ascorbate)/dehydroascorbate (DHA), glutathione (GSH)/glutathione dimer (GSSG) and quinols/quinones and will be the principal focus of investigation in this research program. These have been selected for the following reasons: i. vitamin C and GSH are predominant antioxidants in mammalian systems ii. quinones accounts for a large percentage of all toxic reactions. The latter case is also more complex in that quinol/quinone redox couples are associated with well described antioxidant protective effects. Therefore, the principal long-term objective of this research program is to elucidate the mechanism(s) by which redox recyclable small molecule oxidants such as quinols/quinones influences protein S-thiolation chemistry. This includes protein modifications/thiol-exchange reactions and how these modifications impact redox homeostasis through modulation of enzyme activity and toxic outcome. In order to elucidate antioxidants/oxidant impact on thiol redox chemistry, the following objectives will be performed: 1) Detailed characterization of DHA-mediated S-thiolation by elucidation of DHA-thiol adduct formation and identification of S-thiolated proteins in cells and blood under circumstances of elevated DHA levels. 2) Characterization of GSH exchange reactions with cellular redox proteins and 3) Comprehensive investigation of quinone thiol reactions in cells and with redox proteins that are targets of such reactions. These studies will take advantage of experiments in cells and analytical techniques such as liquid chromatography, mass spectrometry, NMR including proteomics approaches. The anticipated results will give detailed insight into the complexity of S-thiolations and redox regulations. The outcomes will further our knowledge about the interplay between redox couples and protein S-thiolation. This knowledge is anticipated to be a first important step toward developing potential markers and new means to modulate oxidative stress.