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: 2019
• 资助国家: 加拿大
• 起止时间: 2019-01-01 至 2020-12-31
• 项目状态: 已结题
• 来源: https://www.nserc-crsng.gc.ca/ase-oro/Details-Detailles_eng.asp?id=685845
• 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失调中起重要作用。 蛋白质中具有氧化还原活性的半胱氨酸残基含有高度反应性的硫醇，其是活性氧（ROS; O2？_，?哦，H2 O2，？NO，ONOOH）和氧化应激产生的反应性羰基。为了保护它们的完整性，细胞含有与ROS反应以减少其有害影响的清除剂。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。在此过程中，抗坏血酸盐被氧化成脱氢抗坏血酸盐。脱水抗坏血酸（DHA）是一种不稳定的化合物，如果不能迅速回收回抗坏血酸，就会不可逆地降解为反应性羰基化合物，能够在称为抗坏血酸化的过程中修饰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巯基发生特异性反应。然而，关于这种修饰的肽/蛋白质和细胞性质的信息还有待阐明。像AA一样，三肽谷胱甘肽（GSH）达到高细胞内浓度。GSH参与ROS/外源性物质的消除，并参与蛋白S-谷胱甘肽化，这是一种翻译后修饰，旨在保护半胱氨酸巯基免受ROS的侵害。最近，我们证明，当一种蛋白质与GSH孵育一段时间后，只有一小部分蛋白质被S-谷胱甘肽化。然而，在DHA的存在下，丰富的蛋白质S-谷胱甘肽化立即发生，连接AA和GSH的抗氧化活性。基于先前的结果和文献背景，我们统一的假设是蛋白质抗坏血酸化和S-谷胱甘肽化是整合的细胞过程。抗坏血酸的氧化产物脱氢抗坏血酸可以诱导蛋白质S-谷胱甘肽化，然而，脱氢抗坏血酸也可以转化为不可逆地修饰蛋白质的硫醇反应性中间体。 这些过程取决于细胞中脱氢抗坏血酸和谷胱甘肽的水平、氧化应激的性质和程度。* 目标1：蛋白质抗坏血酸化 *A.抗坏血酸化与单个蛋白质的结构表征（短期）*B。细胞中抗坏血酸化的蛋白质靶点的鉴定（中期至长期）* 目的2：蛋白质谷胱甘肽化 *A.开发两种质谱法鉴定S-谷胱甘肽化蛋白（短期）*B。氧化应激诱导的蛋白质谷胱甘肽化：蛋白质靶点的鉴定（中长期）。DHA在体外诱导蛋白S-谷胱甘肽化和蛋白功能（长期）。目的3：蛋白质抗坏血酸化和谷胱甘肽化的关联：定量观点。这项研究计划将允许抗坏血酸化和蛋白S-谷胱甘肽化的蛋白质靶点的鉴定，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，该计划的研究开辟了将以前的发现计划与现在联系起来，以研究药物/代谢物/外源性物质如何通过蛋白质抗坏血酸化和谷胱甘肽化干扰细胞氧化还原电位，将药物不良反应加入细胞氧化还原调节。该研究计划将为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功能作用的理解;抗坏血酸/谷胱甘肽，并领导一系列新型生物标志物的发现。