The dynamics of protein/peptide self-assembly in stress granules

应激颗粒是由细胞中蛋白质和RNA结合而成的。它们被认为在热或氧化应激后形成可逆的集合，以存储部分翻译的RNA和蛋白质，这些RNA和蛋白质可以在恢复后分解。很明显，细胞质和细胞核中的这些非膜结合结构在细胞存活中发挥着重要作用，也可能是亨廷顿病(Huntington‘s，HO)、阿尔茨海默病(AD)和肌萎缩侧索硬化症(AmyoStrategic Always Ssis)等神经退行性疾病中蛋白质错误折叠的先兆。目前还不清楚这些结构是如何组装和分解的，更重要的是，它们如何转化为不可逆转的淀粉样原纤维结构仍然是一个谜。本项目旨在研究低复杂性蛋白质的自组装和结构，如多聚Q和二肽重复蛋白在一系列不同条件下的自组装和结构，以阐明动态组装、凝胶和拆卸可能发生的方式。这将提供关于这些应激颗粒在细胞中的作用以及它们可能如何被调控的有价值的信息。我们假设凝聚体在特定的相空间中形成，它们的性质高度依赖于特定的RNA和蛋白质比例(图I)。凝聚会导致局部环境的形成，与大块液体相比，局部环境会受到明显的扰动。这些变化潜在地提供了特定的环境，其中纤维形成是首选的。目的：1.探索一系列环境(离子强度、pH、多阴离子如RNA)对低复杂结构域蛋白质和多肽的自组装和结构的影响，并建立相图来描述它们的动态范围。阐明了从液体到水凝胶再到固相和相反的相变过程中形成的中间体的结构。破译产生和分解粘结物的时间路径该项目将需要对组装物进行生物物理和电子显微镜表征，并将使用X射线纤维衍射来检查和阐明结构中间体。低温电子显微镜将被用来检查纤维中间体的结构和形态。原子力显微镜(AFM)将与肯特大学的薛伟峰合作进行，薛伟峰是使用原子力显微镜检测淀粉样纤维的专家。这将为相图中不同阶段凝聚体的形态和材料性质提供有价值的信息。博士项目将提供关于凝聚体在体外如何以及在什么条件下形成的详细了解，它们的性质和组成是什么。在这个过程中是否形成淀粉样纤维将被揭开，我们的工作将回答根本的首要问题，即这些结构是否先于神经变性中聚集的病理性蛋白的形成。利用多种方法的强大组合，我们将确定一种伴随着从功能自组装到病理性自组装的“切换”的机制。

Stress granules are formed from the combination of proteins and RNA in cells. They are thought to form reversible assemblies following heat or oxidative stress in order to store partially translated RNA and proteins, which can be disassembled following recovery. It is clear that these non-membrane bound structures in the cytoplasm and nucleus of cells play an important role in cell survival and may also serve as the precursor for protein misfolding in neurodegenerative diseases such as Huntington's (HO) and Alzheimer's disease (AD), and Amyotrophic lateral sclerosis. How these structures assemble and disassemble is currently unclear and importantly, how they are transformed into irreversible amyloid fibril structures remains a mystery. This project aims to examine the self-assembly and structure of low-complexity proteins such as polyQ containing- and dipeptide repeat proteins under a range of different conditions to elucidate the way in which dynamic assembly, gelation and disassembly may take place. This will provide valuable information regarding the role of these stress granules in cells and how they may be regulated. We hypothesise that coacervates form in a certain phase space and that their properties are highly dependent on the specific RNA and protein ratios (Figure I). Coacervation will lead to the formation of local environments that are significantly perturbed compared to the bulk liquid phase. These changes potentially provide specific environments where fibril formation is preferred. Aims:1. Explore the effect of a range of environments (ionic strength, pH, polyanions e.g RNA) on the self-assembly and structure of low complexity domain proteins and peptides and to create a phase diagram to describe their dynamic range.2. Elucidate the structures of intermediates formed during phase transition from liquid to hydrogel to solid phase and in reverse.3. Decipher the temporal pathway that generates and disassembles coacervatesThe project will entail biophysical and electron microscopy characterisation of assemblies and X-ray fibre diffraction will be used to examine and elucidate the structural intermediates. Cryo-Electron microscopy will be used to examine the structure and morphology of fibrous intermediates. Atomic force microscopy (AFM) will be conducted in collaboration with Wei-Feng Xue from the University of Kent who is an expert in the use of AFM for amyloid fibrils. This will provide valuable information regarding the morphology and the materials properties of coacervates at different stages in the Phase diagram. The PhD project will provide a detailed understanding of how and under what conditions coacervates form in vitro, what their properties and composition are. Whether amyloid fibrils are formed during the process will be uncovered and our work will answer the fundamental overarching question of whether these structures precede formation of aggregated, pathological protein in neurodegeneration. Using powerful combination of approaches, we will identify a mechanism that accompanies the "switch" from functional to pathological self-assembly.