The interplay between the UPR and protein biogenesis at the ER

项目摘要/摘要： 分泌蛋白和膜蛋白占人类所有蛋白质的30%左右，它们是共翻译的 跨过内质网或插入内质网。这些新生多肽是折叠的 在内质网中的伴侣和折叠酶的帮助下转化为功能性蛋白质。蛋白质折叠中的缺陷 导致错误折叠的蛋白质积累并触发内质网应激，从而激活未折叠的 蛋白质反应(UPR)。在三种主要的UPR传感器中，IRE1α是最保守的ER局部化传感器 内质网应激时通过寡聚/磷酸化激活的跨膜激酶/核糖核酸酶。一次 IRE1α被激活后，介导XBP1u mRNA的剪接，产生活性转录因子XBP1s， 这驱动了UPR靶基因的表达，以缓解内质网应激。此外，Ire 1α还混杂地切割ER- 通过调节的IRE1依赖的衰变(RIDD)途径定位的mRNAs，以减轻 进入蛋白质负荷。然而，在慢性内质网应激条件下，IRE1α从支持生存 导致细胞死亡，这与包括2型在内的人类疾病有关 糖尿病和癌症。尽管生理上很重要，但控制激活和失活的因素 IRE1α/XBP1信号转导机制尚不清楚。 我们最近发现Ire_1α与Sec61/Sec63转运子复合体形成复合体 获取其信使核糖核酸底物。在目前的供资期间，我们已经表明，Sec61易位桥 IRE1α与SEC63/BIP复合体一起在持续的内质网应激过程中关闭IRE1α信号。我们的研究 发现Sec63/Bip复合体也负责释放堵塞的Sec61转运子以及 促进内质网中蛋白质折叠。这些新的发现提出了一种假设，即IRE1α/Sec61/Sec63 复合体在维持ER的IRE1α/XBP1信号的激活和失活中起核心作用 细胞内的动态平衡。在下一个资助期，我们将通过(I)确定这一因素的作用来检验这一假设 在内质网应激期间做出生死决定的复杂性；(Ii)确定 Ire 1α/Sec61/Sec63/BiP复合体；(Iii)确定该复合体在蛋白质传感/应答中的作用 内质网中的易位缺陷。在一个独立的目标中，我们将在一个 胞质质量控制和IRE1XBP1信号转导。我们计划使用CRISPR/CAS9的组合方法 编辑细胞、生化重建和解决这些问题的结构方法。总体而言，我们 预计这些研究将提供对UPR和蛋白质转运/质量如何的机械性洞察 调控通路共同作用，维持内质网内稳态。从这些研究中获得的知识将 告知对几种人类疾病可能的治疗方法的开发，包括糖尿病、癌症和 多囊性肝病。

Project Summary/Abstract: Secretory and membrane proteins, which account for ~30% of all human proteins, are co-translationally translocated across or inserted into the endoplasmic reticulum (ER). These nascent polypeptides are folded into functional proteins with the help of chaperones and folding enzymes in the ER. Defects in protein folding lead to the accumulation of misfolded proteins and the triggering of ER stress, which activates the unfolded protein response (UPR). Of the three major UPR sensors, IRE1α is the most conserved ER-localized transmembrane kinase/RNase that is activated through oligomerization/phosphorylation upon ER stress. Once activated, IRE1α mediates the splicing of XBP1u mRNA to produce an active transcription factor, XBP1s, which drives expression of UPR target genes to mitigate ER stress. Also, IRE1α promiscuously cleaves ER- localized mRNAs through the regulated Ire1-dependent decay (RIDD) pathway to reduce the burden of the incoming protein load. Under chronic ER stress conditions, however, IRE1α switches from the pro-survival mode to pro-apoptotic mode, resulting in cell death, which is associated with human diseases including, type 2 diabetes and cancer. Despite the physiological importance, the factors that control activation and inactivation of IRE1α/XBP1 signaling remain unclear. We have recently discovered that IRE1α forms a complex with the Sec61/Sec63 translocon complex to access its mRNA substrates. In the current funding period, we have shown that the Sec61 translocon bridges IRE1α with the Sec63/BiP complex to turnoff IRE1α signaling during persistent ER stress. Our studies discovered that the Sec63/BiP complex is also responsible for freeing clogged Sec61 translocons as well as promoting protein folding in the ER. These new findings raise the hypothesis that the IRE1α/Sec61/Sec63 complex plays a central role in the activation and inactivation of IRE1α/XBP1 signaling to maintain ER homeostasis in cells. In the next funding period, we will test this hypothesis by (i) determining the role of this complex in making life-or-death decisions during ER stress; (ii) determining the architecture of the IRE1α/Sec61/Sec63/BiP complex; (iii) determining the role of this complex in sensing/responding to protein translocation defects in the ER. In an independent aim, we will establish a novel functional link between a cytosolic quality control and IRE1α/XBP1 signaling. We plan to use a combined approach of CRISPR/Cas9 edited cells, biochemical reconstitution, and structural approaches to address these problems. Overall, we expect these studies will provide a mechanistic insight into how the UPR and protein translocation/quality control pathways work together to maintain ER homeostasis. The knowledge gained from these studies will inform the development of possible treatments for several human diseases including diabetes, cancer, and polycystic liver diseases.