REGULATION OF IRE1A SIGNALING BY THE SEL1L-HRD1 ERAD COMPLEX

描述(申请人提供)：我们的长期目标是通过细胞中的两个关键质量控制系统--内质网相关降解(ERAD)和未折叠蛋白反应(UPR)--描述维持内质网(ER)动态平衡的分子机制。最近，我们发现了一种新的IRE1α调控因子，它是UPR中最保守的传感器(他等人)。Dev Cell 2012)，并报道了可诱导Sel1L基因敲除(Sel1LIKO)小鼠和细胞模型的产生和特征(Sun等人。PNAS 2014)，这是哺乳动物ERAD中泛素连接酶Hrd1的辅助因子。在这一应用的初步数据中，我们发现了UPR和ERAD之间的一个独特的串扰，即Sel1L-Hrd1ERAD复合体对IRE1ERAD稳定性的调节。在这里，我们发现Sel1L-Hrd1ERAD功能的丧失导致IRE1ERAD蛋白在各种组织和细胞类型中的戏剧性积累，包括胰腺、结肠、脾、脂肪组织、MEF、巨噬细胞等。在没有Sel1L的情况下，IRE1ERAD蛋白的积累不依赖于转录调控，指向转录后控制。事实上，Ire 1α与Sel1L相互作用，并显著稳定在 缺少Sel1L或Hrd1。因此，这些数据表明IRE1ERAD是一个容易错误折叠的Sel1L-Hrd1ERAD底物。在这里，我们建议检验以下假设，即IRE1ERAD是ERAD底物，并且Sel1L-Hrd1ERAD复合体通过调节IRE1ERAD信号的降解来负性调节IRE1ERAD信号的幅度。利用我们为Sel1L ERAD和IRE1ERAD建立的系统和工具，我们将完成以下目标：(1)确定IRE1ERAD对IRE1ERAD信号和细胞生存的生物学意义；(2)确定错误折叠的IRE1α蛋白如何被识别并传递到Sel1L-Hrd1ERAD复合体；以及(3)阐明错误折叠的IRE1ERAD蛋白如何被Sel1L-Hrd1ERAD复合体降解。这项研究的成功完成不仅可能为研究IRE1α和ERAD生物学提供关键的见解，而且还可能揭示IRE1α信号的一种新的调控机制。这项研究将为利用具有重要生理意义的内源性底物研究ERAD的复杂机制提供前所未有的机会，从而对我们的生理性ERAD和UPR生物学观点产生强大的影响。

DESCRIPTION (provided by applicant): Our long-term goal is to delineate the molecular mechanisms underlying the maintenance of endoplasmic reticulum (ER) homeostasis by two key quality-control systems in the cell, ER-associated degradation (ERAD) and unfolded protein response (UPR). Recently, we have identified a novel regulator of IRE1α, the most conserved sensor of the UPR (He et al. Dev Cell 2012), and reported the generation and characterization of inducible Sel1L knockout (Sel1LIKO) mouse and cell models (Sun et al. PNAS 2014), a cofactor of the ubiquitin ligase Hrd1 in mammalian ERAD. In the preliminary data of this application, we discovered a unique crosstalk between UPR and ERAD, namely the regulation of IRE1α stability by the Sel1L-Hrd1 ERAD complex. Here we showed that loss of Sel1L-Hrd1 ERAD function leads to a dramatic accumulation of IRE1α protein in various tissues and cell types including pancreas, colon, spleen, adipose tissue, MEFs, macrophages and etc. IRE1α accumulation in the absence of Sel1L is independent of transcriptional regulation, pointing to a post-transcriptional control. Indeed, IRE1α interacts with Sel1L and is significantly stabilized in the absence of Sel1L or Hrd1. Thus, these data point to IRE1α as a misfolding- prone Sel1L-Hrd1 ERAD substrate. Here we propose to test the hypotheses that IRE1α is an ERAD substrate and that the Sel1L-Hrd1 ERAD complex negatively regulates the amplitude of IRE1α signaling by mediating its degradation. Taking advantage of systems and tools that we have generated for both Sel1L ERAD and IRE1α, we will accomplish the following Aims: (1) Determine the biological significance of IRE1α ERAD on IRE1α signaling and cell survival; (2) Determine how misfolded IRE1α protein is recognized and delivered to the Sel1L-Hrd1 ERAD complex; and (3) Elucidate how misfolded IRE1α protein is degraded by the Sel1L-Hrd1 ERAD complex. Successful completion of this study may not only provide key insights into IRE1α and ERAD biology, but also uncover a novel regulatory mechanism for IRE1α signaling. This study will provide an unprecedented opportunity to investigate the complicated mechanism of ERAD using an endogenous substrate with great physiological significance, thus exerting a powerful influence on our views of physiological ERAD and UPR biology.