New Tools to Measure and Correct Endoplasmic Reticulum Stress in Single Living

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Aided by chaperones and other activities, proteins of the secretory pathway fold to their correct shapes in the endoplasmic reticulum (ER). But, if ER folding capacity is exceeded, unfolded proteins start to aggregate. This imbalanced condition—called ER stress—is being linked to diverse diseases, such as type 2 diabetes and cancer. The unfolded protein response (UPR) can rebalance a stressed ER, but if the stress is too great (or the response too weak), cells appear to cross a “tipping point”, and undergo apoptosis. This could explain why overworked insulin-producing pancreatic β-cells die in type 2 diabetes. On the other hand, a hyperactive UPR may allow malignant cells to survive hostile environments, promoting cancer. If we could measure both ER stress and the strength of corrective responses directly in healthy and diseased cells, we would be able to test these ideas quantitatively, and design rational therapies for these diseases. Structural engineers study system integrity by applying loads, measuring stresses, perturbing reinforcements, and making corrections. We propose similar approaches to ER stress disorders. Protein secretion generates and consumes oxidizing equivalents, so it should be possible to follow deviations in the ER's redox potential from its resting setpoint as an analog measure of ER stress. For this purpose, we are making redox-responsive fluorescent proteins to gauge the ER's redox potential in single, living cells. We have learned to stably perturb ER protein folding by converting an unfolded protein sensor called Ire1 into a finely adjustable rheostat. In living pancreatic β-cells, these tools will allow us to quantify the ER stress caused by insulin folding load, in both healthy and diabetic states. Finally, using a biochemical assay we have developed, we will discover molecules targeting Ire1, to increase or decrease ER folding ability, as appropriate corrective measures for various ER stress disorders.