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

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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.