Protein Ascorbylation and Glutathionylation in Oxidative Stress

氧化应激中的蛋白质抗坏血酸化和谷胱甘肽化

• 批准号: RGPIN-2014-04234
• 负责人: Klarskov, Klaus
• 金额: $ 2.55万
• 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: 2014
• 资助国家: 加拿大
• 起止时间: 2014-01-01 至 2015-12-31
• 项目状态: 已结题
• 来源: https://www.nserc-crsng.gc.ca/ase-oro/Details-Detailles_eng.asp?id=566388
• 关键词: Protein; Ascorbylation; Glutathionylation; Oxidative; Stress

Oxidative stress plays an important role in the deregulation of cellular homeostasis. Redox-active cysteine residues in proteins contain highly reactive thiols, which are major targets of reactive oxygen species (ROS; O2?¯, ?OH, H2O2, ?NO, ONOOH) and reactive carbonyls produced by oxidative stress. To protect their integrity, cells contain scavengers that react with ROS to diminish their harmful effects. Ascorbate and glutathione are among the major scavengers of ROS. Ascorbate (AA) is a powerful reducing agent and by consequence an effective antioxidant. In this process ascorbate is oxidized to dehydroascorbate. Dehydroascorbate (DHA) is an unstable compound, which, if not rapidly recycled back to ascorbate, degrades irreversibly to reactive carbonyl compounds capable of modifying nucleophilic amino acid residues in peptides and proteins in a process known as ascorbylation. One of these compounds reacts specifically with glutathione and protein thiol groups. However, information about what peptides/proteins and the cellular nature of this modification is yet to be elucidated. Like AA, the tripeptide glutathione (GSH) reaches high intracellular concentrations. GSH is involved in the elimination of ROS/xenobiotics and is implicated in protein S-glutathionylation, a post-translational modification, proposed to protect cysteine thiols against ROS. Recently, we demonstrated that when a protein was incubated for an extended period of time with GSH, only a minor fraction of the protein was S-glutathionylated. However, in the presence of DHA abundant protein S-glutathionylation occurs immediately, linking the antioxidant activities of AA and GSH. Based on the previous results and literature background, our unifying hypothesis is that protein ascorbylation and S-glutathionylation are integrated cellular processes. The oxidation product of ascorbate, dehydroascorbate, can induce protein S-glutathionylation, however, dehydroascorbate can also convert to a thiol reactive intermediate that irreversibly modifies proteins. These processes are dependent on the levels of dehydroascorbate and glutathione in the cell, the nature and extent of the oxidative stress. Following objectives will be performed: Objective 1: Protein ascorbylation A. Structural characterization of ascorbylation with individual proteins (short-term) B. Identification of protein targets of ascorbylation in cells (medium to long-term) Objective 2: Protein glutathionylation A. Development of two mass spectrometry approaches for the identification of S-glutathionylated proteins (short-term) B. Oxidative stress-induced protein glutathionylation: identification of protein targets (medium-long term) C. DHA induced protein S-glutathionylation in vitro and protein function (long-term). Objective 3: Association of protein ascorbylation and glutathionylation: a quantitative perspective. This research program will allow identification of protein targets of ascorbylation and protein S-glutathionylation and improve our understanding of the biological role of these post translational modifications in oxidative stress. In the long term, this program’s research opens up for linking the previous discovery program with the present to investigate how drugs/metabolites/xenobiotics interfere with the cellular redox potential through protein ascorbylation and glutathionylations, joining adverse drug reactions to the cellular redox regulation. This research program will provide training of HQP in oxidative stress and state-of-the art analytical chemical approaches to improve our understanding of the functional roles of two major cellular antioxidants; ascorbate/glutathione and lead the discovery of a novel series of biomarkers.

氧化应激在细胞内稳态的破坏中起着重要的作用。蛋白质中具有氧化还原活性的半胱氨酸残基含有高活性的硫醇，这些硫醇是氧化应激产生的活性氧物种(ROS；O2？，？OH，H_2O_2，？NO，ONOOH)和活性羰基的主要靶标。为了保护它们的完整性，细胞中含有清道夫，它们与ROS发生反应，以减少它们的有害影响。抗坏血酸和谷胱甘肽是ROS的主要清除剂。抗坏血酸(AA)是一种强大的还原剂，因此也是一种有效的抗氧化剂。在这个过程中，抗坏血酸被氧化成脱氢抗坏血酸。脱氢抗坏血酸(DHA)是一种不稳定的化合物，如果不能迅速循环回到抗坏血酸，就会不可逆转地降解为活性羰基化合物，能够在一个称为抗坏血酸的过程中修饰多肽和蛋白质中的亲核氨基酸残基。其中一种化合物与谷胱甘肽和蛋白质硫醇基团发生特异性反应。然而，关于什么多肽/蛋白质和这种修饰的细胞性质的信息还没有被阐明。与AA一样，三肽谷胱甘肽(GSH)的细胞内浓度也很高。谷胱甘肽参与清除ROS/外源化合物，并参与蛋白质S谷胱甘肽基化，这是一种翻译后修饰，旨在保护半胱氨酸硫醇免受ROS的影响。最近，我们证明了当蛋白质与谷胱甘肽孵育一段较长的时间时，只有一小部分蛋白质被S谷胱甘肽化。然而，在DHA存在下，富含蛋白质的S-谷胱甘肽立即发生谷胱甘肽基化，将AA和GSH的抗氧化活性联系在一起。基于前人的研究结果和文献背景，我们的统一假设是蛋白质抗坏血酸和S-谷胱甘肽的相互作用是完整的细胞过程。抗坏血酸的氧化产物脱氢抗坏血酸可以诱导蛋白质S谷胱甘肽基化，但脱氢抗坏血酸也可以转化为硫醇反应中间体，不可逆地修饰蛋白质。这些过程取决于细胞中脱氢抗坏血酸和谷胱甘肽的水平，以及氧化应激的性质和程度。将完成以下目标：目标1：蛋白质抗坏血酸作用A.与单个蛋白质抗坏血酸作用的结构特征(短期)B.细胞中抗坏血酸作用的蛋白质靶标的鉴定(中长期)目标2：蛋白质谷胱甘肽基化A.建立两种质谱学方法鉴定S-谷胱甘肽基化蛋白(短期)B.氧化应激诱导的蛋白质谷胱甘肽基化：蛋白质靶标的鉴定(中长期)C.DHA诱导的体外蛋白质S-谷胱甘肽基化和蛋白质功能(长期).目的3：从定量的角度研究蛋白质抗坏血酸和谷胱甘肽的相互作用。这一研究计划将使我们能够识别抗坏血酸化和S谷胱甘肽基化的蛋白质靶标，并提高我们对这些翻译后修饰在氧化应激中的生物学作用的理解。从长远来看，该计划的研究将把以前的发现计划与现在联系起来，调查药物/代谢物/外源生物如何通过蛋白质抗坏血酸和谷胱甘肽基化干扰细胞氧化还原潜力，加入药物对细胞氧化还原调节的不良反应。这项研究计划将为HQP提供氧化应激和最先进的分析化学方法方面的培训，以提高我们对两种主要细胞抗氧化剂：抗坏血酸/谷胱甘肽的功能作用的理解，并引领一系列新生物标志物的发现。