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

在伴侣和其他活动的帮助下，分泌途径折叠的蛋白质 到它们在内质网(ER)的正确形状。但是，如果ER折叠 超出容量时，未折叠的蛋白质开始聚集。这种不平衡 被称为内质网应激的疾病与多种疾病有关，例如2型 糖尿病和癌症。未折叠蛋白反应(UPR)可以重新平衡 应激，但如果压力太大(或反应太弱)，细胞 似乎越过了一个“临界点”，并经历了细胞凋亡。这可以解释为什么 过度劳累产生胰岛素的胰腺β细胞在2型糖尿病中死亡。论 另一方面，高度活跃的UPR可能允许恶性细胞在敌对状态下存活 环境，促进癌症。如果我们能同时测量内质网压力和 直接在健康和患病细胞中的矫正反应的强度，我们将 能够定量地测试这些想法，并为这些想法设计合理的治疗方法 疾病。 结构工程师通过施加载荷、测量 强调，扰乱增援，并进行纠正。我们提出了类似的建议 内质网应激障碍的治疗方法。蛋白质分泌产生并消耗 氧化当量，所以应该可以跟踪ER的偏差 从静息设定点的氧化还原电位作为内质网应激的模拟测量。为 为此，我们正在制造氧化还原反应荧光蛋白来测量 内质网在单个活细胞中的氧化还原电位。我们已经学会了稳定地扰乱ER 通过将名为IRE1的未折叠蛋白质传感器转换为精细的 可调式变阻器。在活的胰腺β细胞中，这些工具将允许我们 量化健康人和糖尿病人因胰岛素折叠负荷引起的内质网应激 各州。最后，使用我们开发的生化分析方法，我们将发现 靶向IRE1的分子，以增加或降低ER折叠能力，如 针对各种内质网应激障碍采取适当的纠正措施。

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.