Protein S-thiolations/trans-thiolations and oxidative stress

蛋白质 S-硫醇化/反式硫醇化和氧化应激

• 批准号: RGPIN-2020-05786
• 负责人: Klarskov, Klaus
• 金额: $ 2.33万
• 依托单位: Université de Sherbrooke
• 依托单位国家: 加拿大
• 项目类别: Discovery Grants Program - Individual
• 财政年份: 2021
• 资助国家: 加拿大
• 起止时间: 2021-01-01 至 2022-12-31
• 项目状态: 已结题
• 来源: https://www.nserc-crsng.gc.ca/ase-oro/Details-Detailles_eng.asp?id=740828
• 关键词: Protein; thiolations; trans; oxidative; stress

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.

过度形成的活性氧（ROS）不能通过细胞氧化还原防御机制来缓解，从而导致氧化应激。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，衰老和糖尿病和癌症等主要疾病。生物体已经发展出复杂的生化途径来控制ROS水平并减轻潜在的损害。其中包括肽/蛋白硫醇氧化还原化学、抗氧化蛋白和丰富的小内、外源性抗氧化氧化还原偶对。我们对这些低分子量氧化还原偶对如何调节蛋白质s -硫醇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来对抗不受调节的ROS形成非常重要。三个基本的硫醇相互作用的氧化还原对是；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）和醌类化合物将是本研究项目的主要研究重点。选择这些药物的原因如下：1 .维生素C和谷胱甘肽是哺乳动物系统中主要的抗氧化剂。醌类物质在所有毒性反应中占很大比例。后一种情况也更为复杂，因为喹啉/醌氧化还原对具有良好的抗氧化保护作用。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（如喹啉/醌类）影响蛋白质s -硫代化化学的机制。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)在DHA水平升高的情况下，通过阐明DHA-硫醇加合物的形成和鉴定细胞和血液中s -硫醇化蛋白，详细表征DHA介导的s -硫醇化。2)表征谷胱甘肽与细胞氧化还原蛋白的交换反应；3)全面研究细胞内醌硫醇反应及其靶氧化还原蛋白的交换反应。这些研究将利用细胞实验和分析技术，如液相色谱，质谱，核磁共振包括蛋白质组学方法。预期的结果将为s -硫代化合物和氧化还原调控的复杂性提供详细的见解。这些结果将进一步加深我们对氧化还原偶对与蛋白质s -硫基化之间相互作用的认识。这一知识有望成为开发潜在标记物和调节氧化应激新手段的重要的第一步。