REGULATION OF IRE1A SIGNALING BY THE SEL1L-HRD1 ERAD COMPLEX

描述（由申请人提供）：我们的长期目标是通过细胞中两个关键的质量控制系统（ER相关降解（ERAD）和未折叠蛋白反应（UPR））来描述维持内质网（ER）稳态的分子机制。最近，我们已经鉴定了IRE 1 α的一种新型调节因子，IRE 1 α是UPR最保守的传感器（He et al. Dev Cell 2012），并报道了诱导型Sel 1 L敲除（Sel 1 LIKO）小鼠和细胞模型的产生和表征（Sun et al. PNAS 2014），哺乳动物ERAD中泛素连接酶Hrd 1的辅因子。在本申请的初步数据中，我们发现了UPR和ERAD之间的独特串扰，即Sel 1 L-Hrd 1 ERAD复合物对IRE 1 α稳定性的调节。我们发现Sel 1 L-Hrd 1 ERAD功能缺失导致IRE 1 α蛋白在胰腺、结肠、脾脏、脂肪组织、MEFs、巨噬细胞等多种组织和细胞中大量积累。事实上，IRE 1 α与Sel 1 L相互作用，并在 Sel 1 L或Hrd 1的缺失。因此，这些数据表明IRE 1 α是一种易于错误折叠的Sel 1 L-Hrd 1 ERAD底物。在这里，我们提出测试的假设，IRE 1 α是ERAD底物和Sel 1 L-Hrd 1 ERAD复合物负调控IRE 1 α信号的幅度通过介导其降解。利用我们已经开发的Sel 1 LERAD和IRE 1 α的系统和工具，我们将完成以下目标：（1）确定IRE 1 α ERAD对IRE 1 α信号传导和细胞存活的生物学意义;（2）确定错误折叠的IRE 1 α蛋白如何被识别并递送到Sel 1 L-Hrd 1 ERAD复合物;（3）阐明Sel 1 L-Hrd 1 ERAD复合物对错误折叠的IRE 1 α蛋白的降解作用。这项研究的成功完成不仅可以为IRE 1 α和ERAD生物学提供关键见解，而且还可以揭示IRE 1 α信号转导的新调控机制。本研究为利用具有重要生理意义的内源性底物研究ERAD的复杂机制提供了前所未有的机会，从而对我们对生理ERAD和UPR生物学的看法产生了强大的影响。

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.