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

应激颗粒是由细胞中的蛋白质和RNA结合形成的。它们被认为在热或氧化应激后形成可逆的组装体，以储存部分翻译的RNA和蛋白质，这些RNA和蛋白质可以在恢复后分解。很明显，细胞的细胞质和细胞核中的这些非膜结合结构在细胞存活中起重要作用，并且还可以作为神经变性疾病如亨廷顿病（HO）和阿尔茨海默病（AD）以及肌萎缩性侧索硬化症中蛋白质错误折叠的前体。这些结构如何组装和分解目前尚不清楚，重要的是，它们如何转化为不可逆的淀粉样纤维结构仍然是一个谜。该项目旨在研究低复杂性蛋白质的自组装和结构，例如在一系列不同条件下含polyQ和二肽重复蛋白质，以阐明动态组装，凝胶化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如何被调节的有价值的信息。我们假设凝聚体在一定的相空间中形成，并且它们的性质高度依赖于特定的RNA和蛋白质比例（图I）。凝聚将导致局部环境的形成，与本体液相相比，局部环境被显著扰动。这些变化潜在地提供了其中优选原纤维形成的特定环境。目的：1.探索一系列环境（离子强度、pH、聚阴离子如RNA）对低复杂结构域蛋白质和肽的自组装和结构的影响，并创建相图来描述它们的动态范围.阐明了液相-水凝胶-固相以及液相-水凝胶-固相相变过程中形成的中间产物的结构.该项目将需要生物物理和电子显微镜表征的组件和X射线纤维衍射将用于检查和阐明结构中间体。冷冻电子显微镜将用于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AFM研究淀粉样纤维的专家。这将提供有价值的信息，形态和材料特性的凝聚在不同阶段的相图。博士项目将提供如何以及在什么条件下凝聚体在体外形成，它们的属性和组成是什么详细的了解。淀粉样纤维是否在此过程中形成将被揭示，我们的工作将回答这些结构是否先于神经变性中聚集的病理性蛋白质形成的根本性问题。使用强大的组合的方法，我们将确定一个机制，伴随着“开关”从功能到病理的自组装。

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.