Reactive oxygen species (ROS) perform dual roles in cells, acting as both destructive and constructive agents. At high levels, intracellular ROS facilitate irreversible macromolecular damage and are associated with a range of disease pathologies. At lower physiological levels, ROS play important roles in the activation of beneficial signaling events through the reversible post-translational modification of cysteine and methionine residues. Despite the emerging appreciation for ROS as constructive signaling agents, and the importance for functional redox signaling in managing cellular ROS, relatively few redox-signaling pathways have been characterized. Many proteins susceptible to oxidation have been cataloged. However, the consequences of oxidation and the physiological outcomes have been established for only a limited number of these targets. We argue this central knowledge gap limits larger efforts towards the development of effective therapeutics to help manage cellular ROS. Since its inception, our research program has focused on broadening our knowledge of ROS-based signaling events. Our overarching goal is to elucidate individual pathways activated by the modification of protein cysteine and/or methionine by ROS. Our intent is to uncover and to characterize redox-signaling components, including ROS sources, redox targets, and oxidation regulators, as well as the consequences (outcomes) for signaling at the protein and physiological level. Our research efforts center on two focus areas: (1) an analysis of the role for cysteine oxidation in managing ROS within the endoplasmic reticulum (ER) and (2) the study of the consequences of protein methionine oxidation (MetO) formation and reduction. Our prior work established that oxidation of a conserved cysteine in the Hsp70 BiP alters its chaperone activity to sustain ER function under elevated ROS conditions. Our ongoing work intends to broaden our understanding of redox signaling at the ER and BiP oxidation, answering the questions: What are the local endogenous sources of ROS in the ER that are sensed by BiP? How does BiP oxidation influence known ER stress response pathways? In addition, we intend to bolster the fundamental understanding of MetO formation and resolution in cells, focusing in parallel on what we consider the most prominent gaps in our knowledge of MetO: What are the physiological targets of MetO? What is the role for methionine sulfoxide reductases in regulating individual protein MetO events throughout the cell? By answering these questions, we intend to provide insight into the basic cell functions used to manage cellular ROS and avert cellular damage. We anticipate that our experience and expertise, coupled with a diversity of personnel and scientific approaches, will allow us to make sustained research progress over this MIRA award.