Dynamic Gene Circuit Mapping of Unfolded Protein Response in Type 2 Diabetes

项目摘要/摘要 2型糖尿病给世界带来了巨大的且不断增长的健康负担，有4.4亿病例 据预测，到2030年，全球都会出现这种情况。在2型糖尿病中，增加胰岛素抵抗需要更多的 胰岛β细胞产生更多的胰岛素。由此产生的对蛋白质折叠机制的压力 β细胞触发未折叠蛋白反应(UPR)，这是一个细胞信号网络，作用于修复 在细胞压力面前的动态平衡。然而，未能恢复细胞内稳态的慢性UPR可以 驱使细胞死亡。其结果是形成了一个前馈循环，对剩余的胰岛β细胞造成了更大的压力 并最终导致全身葡萄糖动态平衡的丧失，导致肾脏终末器官损伤， 眼睛、心脏和外周神经系统。 目前的项目提案旨在提高我们可视化和表征基因网络的能力 潜在的UPR调控以及支持生存和支持细胞死亡的途径之间的转换。UPR激活 从三个应激分子传感器开始：Ire 1、、PERK和ATF6。在慢性UPR下，转录 CHOP因子上调，激活下游通路，导致细胞死亡。目标1 拟议的项目使用新的带有荧光的细胞系来表征UPR途径激活的动力学 普遍定期审议组成部分IRE1、PERK、ATF6和CHOP的记者。这些记者将使 在单个细胞水平上观察处于UPR状态的开关。这个系统应该提供一种新的工具来 在细胞应激和2型糖尿病领域，它也可以在未来用于药物筛选 影响UPR途径特定手臂的药物。 理解和治疗2型糖尿病等疾病的一个关键挑战是它们的复杂性， 多个细胞通路之间的串扰。目标2将识别调节UPR的基因组合 胰岛β细胞，保护细胞免受细胞应激的毒性影响，使用一种新型的，高通量的 最近开发的研究基因组合表型效应的方法(CombiGEM)。这 组合遗传方法可能更好地模拟UPR调控及其与其他关键调控因素的相互作用 细胞通路。CombiGEM在永生化β细胞系中确定的候选相互作用将得到验证 在原代小鼠的β细胞中。最终，这些研究可能为UPR在类型上的调制提供洞察力 2糖尿病，具有潜在的治疗应用。

PROJECT SUMMARY/ABSTRACT Type 2 diabetes places a significant and growing health burden on the world, with 440 million cases predicted by the year 2030 worldwide. In type 2 diabetes, increasing insulin resistance demands greater and greater insulin production from pancreatic beta cells. The resulting stress on the protein folding machinery of the beta cells triggers the unfolded protein response (UPR), a cell signaling network that acts to restore homeostasis in the face of cell stress. However, chronic UPR that fails to restore cell homeostasis can instead drive cell death. The result is a feed-forward loop with increased stress on the remaining pancreatic beta cells and eventual loss of systemic glucose homeostasis, leading to devastating end-organ damage in the kidneys, eyes, heart, and peripheral nervous system. The current project proposal aims to improve our ability to visualize and characterize the gene networks underlying UPR regulation and the switch between pro-survival and pro-cell death pathways. UPR activation starts with three molecular sensors of stress: IRE1, PERK, and ATF6. Under chronic UPR, the transcription factor CHOP is upregulated, and it activates downstream pathways that lead to cell death. Aim 1 of the proposed project characterize the dynamics of UPR pathway activation using new cell lines with fluorescent reporters for the UPR components IRE1, PERK, ATF6, and CHOP. These reporters will enable the observation of switches in UPR states in at the level of individual cells. This system should offer a new tool to the fields of cell stress and type 2 diabetes, and it could also be used in the future to screen for pharmaceutical agents that affect specific arms of the UPR pathway. A key challenge to understanding and treating diseases like type 2 diabetes is their complexity, with crosstalk among multiple cell pathways. Aim 2 will identify combinations of genes that modulate the UPR in pancreatic beta cells and that protect cells from the toxic effects of cell stress, using a novel, high-throughput approach recently developed for investigating the phenotypic effects of gene combinations (CombiGEM). This combinatorial genetic approach may better model UPR regulation and its interactions with other key regulatory cell pathways. Candidate interactions identified by CombiGEM in immortalized beta cell lines will be validated in primary murine beta cells. Ultimately, these studies may offer insight into the modulation of the UPR in type 2 diabetes, with potential therapeutic applications.