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物种(ROS)的过度形成会导致氧化应激。这种情况与多种重要的病理生物学状态有关，如细胞/器官毒性、衰老以及糖尿病和癌症等重大疾病。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，抗氧化蛋白质和丰富的小的内源和外源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-硫醇化学的了解有限。深入了解它们与硫醇的详细反应对于了解作用机制(S)、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来对抗放松调节的ROS形成是很重要的。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)和对苯二酚/对苯二酚是三种基本的硫醇相互作用的氧化还原对，将是本研究计划的主要研究重点。选择这些抗氧化剂的原因如下：1.维生素C和GSH是哺乳动物体内的主要抗氧化剂2。喹诺酮类药物在所有毒性反应中占很大比例。后一种情况也更加复杂，因为苯酚/苯醌氧化还原对与众所周知的抗氧化保护作用有关。因此，本研究计划的主要长期目标是阐明氧化还原可回收小分子氧化剂(如苯二酚/苯二酚)通过其影响蛋白质S-硫代化化学的机制(S)。这包括蛋白质修饰/硫醇交换反应，以及这些修饰如何通过调节酶活性和毒性结果来影响氧化还原动态平衡。为了阐明抗氧化剂/氧化剂对硫醇氧化还原化学的影响，我们将进行以下工作：1)在DHA水平升高的情况下，通过阐明DHA-硫醇加合物的形成和细胞和血液中S-硫醇化蛋白的鉴定，详细表征DHA介导的S-硫代化反应。2)GSH与细胞氧化还原蛋白的交换反应的表征；3)细胞中的醌-硫醇反应以及与氧化还原蛋白的反应的综合研究。这些研究将利用细胞实验和分析技术，如液相色谱、质谱学、核磁共振，包括蛋白质组学方法。预期的结果将使人们对S-硫代化和氧化还原法规的复杂性有详细的了解。这些结果将进一步加深我们对氧化还原对和蛋白质S硫基化之间相互作用的了解。这一知识有望成为朝着开发潜在标记物和调节氧化应激的新手段迈出的重要的第一步。

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.