UNRAVELLING THE PROTEIN-LEVEL REGULATION OF ORGANISMS UNDER STRESS

Biological stress is often manifested as changes in reactive oxygen species generation that can be rapidly proliferated to facilitate intracellular signaling. This signaling is frequently accomplished via the reversible post-translational oxidation of cysteine residues, through which oxidation infers site-specific regulation of protein activity and function. Oxidative signaling is implicated in nearly every biological pathway across all taxa, though its precise function and protein targets are largely uncharacterized. The goals of this dissertation are to better characterize stress-related redox pathways in the model green alga, Chlamydomonas reinhardtii, as well as the model tardigrade species, Hypsibius exemplaris. First, the ability of proteomics to increase understanding of stress pathways in plant and algae was thoroughly reviewed (Chapter 2). Oxidized resin assisted capture was then implemented to explore the correlation of oxidative signaling on nitrogen-depletion induced physiological responses in C. reinhardtii (Chapter 3). To increase throughput of the analysis of the redox proteome, a multiplexed method for simultaneous quantification of reduced cysteines, reversibly oxidized cysteines, and the global proteome was developed (Chapter 4). Crosslinking mass spectrometry was then optimized to assess the protein-protein interactions in C. reinhardtii to aid in future studies of protein complex-dependent stress pathways (Chapter 5)Tardigrades are an emerging model system for stress research and therefore required thorough method development for tardigrade husbandry, cryptobiotic characterization, protein extraction, and proteomic analysis (Chapter 6). Finally, these developed methods were implemented to explore the ROS-dependence of tardigrade survival via cryptobiosis (Chapter 7).As demonstrated, this dissertation implements a variety or proteomic methods to describe stress pathways in C. reinhardtii and H. exemplaris, with a sustained focus on the integration of oxidative signaling for organism survival. This work ultimately helps to further characterize the role of reactive oxygen species in cellular stress response and homeostasis.