The interplay between the UPR and protein biogenesis at the ER

UPR 和 ER 蛋白质生物发生之间的相互作用

• 批准号: 10211808
• 负责人: MALAIYALAM MARIAPPAN
• 金额: $ 34.51万
• 依托单位: YALE UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2016
• 资助国家: 美国
• 起止时间: 2016-06-01 至 2025-04-30
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/10211808
• 关键词: ATP phosphohydrolase; Address; Antibodies; Apoptotic; Architecture; Attenuated; Beta Cell; Biochemical; Biogenesis; CRISPR/Cas technology; Carrier Proteins; Cell Death; Cells; Cessation of life; Chronic; Cleaved cell; Complex; Data; Defect; Degradation Pathway; Development; Diabetes Mellitus; Disease; Dominant-Negative Mutation; Electron Transport Complex III; Endoplasmic Reticulum; Enzymes; Funding; Genes; Homeostasis; Hormones; Human; Knowledge; Lead; Life; Link; Malignant Neoplasms; Mediating; Membrane Proteins; Messenger RNA; Molecular; Molecular Chaperones; Monitor; Mutation; Non-Insulin-Dependent Diabetes Mellitus; Pathway interactions; Phosphorylation; Phosphotransferases; Physiological; Play; Protein translocation; Proteins; Quality Control; RNA Splicing; Ribonucleases; Ribosomes; Role; Signal Transduction; Structure; Testing; Ubiquitination; Work; XBP1 gene; endoplasmic reticulum stress; human disease; insight; misfolded protein; novel; polycystic liver disease; polypeptide; prevent; protein folding; reconstitution; recruit; response; secretory protein; sensor; transcription factor

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.

项目概要/摘要： 分泌蛋白和膜蛋白约占人类蛋白质总量的30%， 易位穿过或插入内质网（ER）。这些新生的多肽被折叠 在伴侣蛋白和内质网折叠酶的帮助下转化为功能蛋白。蛋白质折叠缺陷 导致错误折叠的蛋白质的积累和触发ER应激，其激活未折叠的 蛋白质反应（UPR）。在三种主要的UPR传感器中，IRE 1 α是最保守的ER定位的 在ER应激时通过寡聚化/磷酸化激活的跨膜激酶/RNA酶。一旦 激活后，IRE 1 α介导XBP 1u mRNA的剪接，产生活性转录因子XBP 1 s， 其驱动UPR靶基因的表达以减轻ER应激。此外，IRE 1 α混杂地切割ER- 通过受调节的Ire 1依赖性衰变（RIDD）途径定位mRNA， 输入的蛋白质负荷。然而，在慢性内质网应激条件下，IRE 1 α从促存活的 模式转变为促凋亡模式，导致细胞死亡，这与人类疾病相关，包括2型 糖尿病和癌症尽管在生理上很重要，但控制激活和失活的因素 IRE 1 α/XBP 1信号通路的作用机制尚不清楚。 我们最近发现IRE 1 α与Sec 61/Sec 63易位子复合物形成复合物， 获取其mRNA底物。在当前的资助期内，我们已经证明Sec 61易位桥 IRE 1 α与Sec 63/BiP复合物在持续ER应激期间关闭IRE 1 α信号传导。我们的研究 发现Sec 63/BiP复合物也负责释放堵塞的Sec 61 translocons， 促进ER中的蛋白质折叠。这些新发现提出了IRE 1 α/Sec 61/Sec 63 复合物在IRE 1 α/XBP 1信号通路的激活和失活中发挥重要作用，以维持ER 细胞内的稳态在下一个融资期，我们将通过以下方式检验这一假设：（i）确定这一假设的作用， 复杂的，在作出生死决定，在ER压力;（ii）确定的架构， IRE 1 α/Sec 61/Sec 63/BiP复合物;（iii）确定该复合物在感测/响应蛋白质中的作用 ER中的易位缺陷。在一个独立的目标，我们将建立一个新的功能之间的联系， 胞质质量控制和IRE 1 α/XBP 1信号传导。我们计划使用CRISPR/Cas9的组合方法， 编辑的细胞，生化重建和结构方法来解决这些问题。总的来说，我们 预计这些研究将提供一个机制的洞察如何UPR和蛋白质易位/质量 控制通路共同作用以维持ER稳态。从这些研究中获得的知识将 为几种人类疾病的可能治疗方法的发展提供信息，包括糖尿病，癌症， 多囊性肝病