Exploring NADPH Levels during ER Stress using Novel HomoFRET Sensors

In beta-cells, insulin misfolding occurs at rates as high as 20%. Misfolded insulin aggregates lead to endoplasmic reticulum (ER) stress and the unfolded protein response (UPR). The adaptive UPR works to refold insulin, but if unsuccessful leads to programmed cell death. Proper insulin refolding requires reducing equivalents, which the ER lacks, thus external support in the form of NADPH is needed. This thesis investigates the role of NADPH in supporting insulin refolding in the ER. A novel homoFRET reporter, Apollo-IRE1, was developed to monitor the progression of ER stress. Apollo-IRE1 functionality was validated with chemical ER stressors. Additionally, our lab’s reporter, Apollo-NADP+, was used to track organelle NADPH levels during ER stress. Mitochondrial NADPH, not cytoplasmic NADPH, was found to be consumed during ER stress. The consumption of mitochondrial NADPH during ER stress demonstrates a potential supportive role of NADPH in the beta-cells protein refolding process.%%%%M.A.S.