Protein Ascorbylation and Glutathionylation in Oxidative Stress

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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 ascorbylationA. 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 glutathionylationA. 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.