REGULATION OF IRE1A SIGNALING BY THE SEL1L-HRD1 ERAD COMPLEX

描述（由申请人提供）：我们的长期目标是描述细胞中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）——维持内质网（ER）稳态的分子机制。最近，我们已经确定了IRE1α的一个新的调节因子，IRE1α是UPR中最保守的传感器（He等）。Dev Cell 2012)，并报道了诱导Sel1L基因敲除（Sel1LIKO）小鼠和细胞模型的生成和特性（Sun等）。PNAS 2014)，哺乳动物ERAD中泛素连接酶Hrd1的辅助因子。在本应用的初步数据中，我们发现了UPR和ERAD之间独特的串扰，即Sel1L-Hrd1 ERAD复合物对IRE1α稳定性的调控。我们发现Sel1L-Hrd1 ERAD功能的缺失导致IRE1α蛋白在胰腺、结肠、脾脏、脂肪组织、mef、巨噬细胞等多种组织和细胞类型中大量积累。在缺乏Sel1L的情况下，IRE1α的积累独立于转录调控，表明存在转录后调控。事实上，IRE1α与Sel1L相互作用，并在

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.