BIORAD ChemiDoc MP imaging system

摘要/摘要 绝大多数分泌和质膜蛋白都是在膜结合的基础上合成的。 核糖体位于内质网(ER)。合成这一子集蛋白质的核糖体的目标是 ER膜由信号识别颗粒(SRP)组成。SRP在信号序列出现时绑定到信号序列 核糖体作为不断增长的多肽链的一部分。然后SRP将新生的多肽交给 Sec61转运子进入或穿过内质网双分子膜。内质网管腔内的蛋白质折叠 以共同翻译的方式进行收益。在蛋白质错误折叠的情况下，一个信号网络，“未折叠的蛋白质” 响应(UPR)“被激活，使内质网的蛋白质折叠能力与进入的蛋白质负荷重新平衡 急诊室。IRE1是UPR的主要蛋白质错误折叠传感器之一。它是一种双功能的激酶/核糖核酸酶，具有 由未折叠的蛋白质结合激活的管腔结构域。它通过一种非常规的信使核糖核酸 剪接反应，利用其胞质核糖核酸酶结构域从编码 转录因子XBP1。XBP1促进一系列伴侣蛋白、蛋白质折叠因子和 蛋白质运输和降解机制的组成部分，以重建动态平衡。IRE1也会降级 选择内质网结合的mRNAs，从而减少内质网蛋白折叠负荷的反应被称为“调节的IRE1 依赖衰变(RIDD)“。IRE1可以直接与共翻译易位机制相互作用，包括 核糖体、SRP和Sec61易位。这些数据(和支持证据)表明，至少在 部分，在与内质网结合的核糖体的物理接触中发挥其功能。然而，当激活时，IRE1会形成 大的低聚团簇。我们最近使用相关的光和电子技术在完整的细胞中可视化了这些簇 显微镜。我们的高分辨率低温断层图像表明，IRE1星系团是由 细小的、解剖的ER管。令人惊讶的是，这些簇完全没有膜结合的核糖体。 这提出了一个悖论，表明活跃的IRE1必须存在于至少两个子群体中，其中一个由 可与核糖体和/或SRP结合的小寡聚体，另一种由不含核糖体的大分子IRE1组成 集群。我们将把IRE1的S靶基因和机械性角色映射到这些状态。具体来说，我们会：(1) 剖析了IRE1选择底物的机理。(2)测定IRE1的原子结构 使用单颗粒冷冻-EM与核糖体、转运子和/或SRP复合。(3)识别亚细胞 IRE1剪接和RIDD裂解的mRNAs的发源地。(4)组装定义的化学计量比的IRE1簇 在体外和在细胞内，并使用生化和基于细胞的分析来评估功能输出。蛋白质平衡的缺陷 处于不断扩大的疾病清单的核心。拟议中的实验旨在定义哺乳动物是如何 IRE1选择其信使核糖核酸靶标，以及在其功能状态下与哪些关键分子相关。这个 这项工作不仅将加深对IRE1细胞生物学的理解，还有望打开新的 在癌症、神经变性等疾病中的临床应用途径。

Abstract / Summary The vast majority of all secreted and plasma membrane proteins are synthesized on membrane-bound ribosomes at the endoplasmic reticulum (ER). Ribosomes synthesizing this subset of proteins are targeted to the ER membrane by the signal recognition particle (SRP). SRP binds to signal sequences as they emerge from the ribosome as part of the growing polypeptide chain. SRP then hands over the nascent polypeptide to the Sec61 translocon for movement into or across the ER membrane bilayer. Protein folding in the ER lumen proceeds co-translationally. In the event of protein misfolding, a signaling network, the “unfolded protein response (UPR)”, is activated that rebalances the ER’s protein folding capacity with the load of proteins entering the ER. IRE1 is one of the UPR’s main protein misfolding sensors. It is a bifunctional kinase/RNase with a lumenal domain that is activated by unfolded protein binding. It signals through a non-conventional mRNA splicing reaction, utilizing its cytoplasmic RNase domain to excise an intron from the mRNA encoding the transcription factor XBP1. XBP1 drives expression of a host of chaperones, protein folding factors, and component of the protein transport and degradation machinery to reestablish homeostasis. IRE1 also degrades select ER-bound mRNAs, thereby reducing the ER protein-folding load in a reaction termed “regulated IRE1 dependent decay (RIDD)”. IRE1 can directly interact with the co-translational translocation machinery, including the ribosome, SRP, and Sec61 translocon. These data (and supporting evidence) suggest that IRE1, at least in part, performs its functions in physical contact with ER-bound ribosomes. When activated, however, IRE1 forms large oligomeric clusters. We recently visualized these clusters in intact cells using correlative light and electron microscopy. Our high-resolution cryo-tomograms demonstrate that IRE1 clusters are convoluted networks of narrow, anatomosing ER tubes. Surprisingly, these clusters are entirely devoid of membrane-bound ribosomes. This poses a paradox, suggesting that active IRE1 must exist in at least two sub-populations, one composed of small oligomers that can ribosome- and/or SRP-associate, the other composed of large ribosome-free IRE1 clusters. We will map IRE1’s target mRNAs and mechanistic roles to these states. Specifically, we will: (1) Dissect the mechanistic principles of substrate selection by IRE1. (2) Determine the atomic structure of IRE1 complexed to ribosomes, translocon, and/or SRP using single-particle cryo-EM. (3) Identify the sub-cellular birthplaces of IRE1-spliced and RIDD-cleaved mRNAs. (4) Assemble IRE1 cluster of defined stoichiometry in vitro and in cells and assess functional outputs with biochemical and cell-based assays. Defects in proteostasis lie at the heart of an ever-expanding list of diseases. The proposed experiments aim to define how mammalian IRE1 selects its mRNA targets and what key molecular players it associates with in its functional states. The work will not only lead to a deeper understanding of the cell biology of IRE1 but also promises to open new avenues for clinical application in cancer, neurodegeneration and other diseases.