Excessive production of intracellular reactive oxygen species (ROS) or oxidative stress is implicated in numerous human diseases such as cancer, diabetes, neurodegenerative diseases and aging. Redox-dependent signaling is a prototypic means of cellular regulation under physiological and pathological conditions. Molecular level studies of redox biology are an increasingly promising direction for revealing novel insights into diseases and identifying novel therapeutic targets.
Our research focuses on developing novel chemical proteomics approaches for enabling functional measurements relevant to redox biology. Specifically, we focus on reversible cysteine modifications, a primary target for ROS, and their functional roles regarding human health and diseases. In this presentation, I would like to highlight our recent advances in developing and optimization of redox proteomics toolsets with emphasis on protein S-glutathionylation (SSG), an important type of reversible cysteine modification by forming protein disulfides with glutathione. To highlight its application, we utilized nanoparticle induced immunosuppression in macrophages as a model system where we hypothesized that SSG is a regulatory mechanism by which engineered nanoparticles (ENPs) may alter macrophage innate immune functions. With the quantitative redox measurements, we reveal that the levels of SSG alterations correlate well with the overall level of cellular ROS and the impairment of macrophage phagocytic function (i.e., CoO > Fe3O4 > SiO2). Furthermore, our data revealed pathway-specific differences between low ROS versus high ROS conditions. Our results for the first time revealed the regulatory molecular insights of SSG in ER stress response and phagocytosis.
To further facilitate the identification of functional targets (Cys sites) or protein networks, we pursued stoichiometric level quantification of SSG occupancy and redox status for protein cysteines in macrophages. Interestingly, proteomic measurements of both dynamic change and stoichiometry for cysteine modification provided valuable insights on redox regulation in subcellular compartments and enabled prediction of specific pathways associated with activation and inhibition of various biological processes. In addition, a number of function relevant proteins and their cysteine sites have been identified as the candidates for further targeted exploration.
Collectively, redox proteomics provides prospective toolset for quantitative studies of redox biology, which facilitates global discovery of novel functional targets, protein network and pathways. Subsequent more accurate targeted quantification for specific protein sets should be powerful for identifying and validating functional targets.