Grant: Discovery Grants Program - Individual

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.