Redox regulation of the UPR sensor Ire1

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Project Summary/Abstract The oxidative protein folding machinery in the endoplasmic reticulum (ER) is currently appreciated as a potentially significant source of reactive oxygen species (ROS). Yet, few specific mechanisms have been described as to how cells manage ROS production at the ER and/or adapt to an accumulation of ER ROS. With little understanding of the pathways for ER ROS management in cells, how dysregulation of ER ROS levels contributes to disease is under-explored. The research goal of this proposal is to elucidate how cells use ROS generated in the ER lumen to initiate signals that promote healthy ROS levels and allow survival through conditions of oxidative ER stress. In order to dissect the molecular mechanisms specific to the production of ER ROS, the Sevier Lab takes advantage of a mutant of the conserved enzyme Ero1 (Ero1*) as an innovative way to induce a bolus of H2O2 within the ER lumen of S. cerevisiae. Using Ero1* to induce ROS within the ER, I uncovered a role for the UPR sensor Ire1 in sensing ER ROS. Guided by the discovery in C. elegans of a redox switch in the kinase domain of IRE-1 that can be activated by cytoplasmic ROS, I have established that genetically blocking Ire1 oxidation at the conserved cysteine (Ire1-C832S) results in enhanced Ire1 enzymatic activity during Ero1* treatment, consistent with UPR dampening as a consequence of C832 oxidation. Based on these data, I hypothesize that ROS generated in the ER lumen during protein folding can oxidize Ire1 C832 in the cytoplasm and initiate an antioxidant response that allows cells to adapt to the unique stress of excess ER ROS. To test this hypothesis, in Aim 1 I will determine the oxidation status of Ire1 when Ero1* over-expression generates a buildup of H2O2 in the ER lumen, and I will show that Ire1 modification occurs at C832. Additionally, I will establish that it is the movement of H2O2 from the ER lumen into the cytosol that facilitates Ire1 modification. In Aim 2, I will assess whether the loss in canonical Ire1 activation is associated with a gain of a new function in antioxidant signaling. My proposed research project is coupled to a broader training plan that will ensure success as I work toward becoming a research group leader. I identified mentoring as a complex professional skill I want to improve. My Sponsors and I have outlined a training plan that will both strengthen my technical and communication skills and also hone my proficiency as a mentor. Specifically, I put forward a plan to integrate the characterization of the biochemical effect(s) of Ire1 oxidation with the mentoring of an undergraduate student that will complete a straightforward analysis of the impact for Ire1 oxidation on Ire1 kinase and RNase activities. I believe the support of my individual Sponsors nestled within the intellectually exciting environment of the Cornell University Biochemistry, Molecular, and Cell Biology graduate program primes me for personal scientific and professional successes.