Formation of membraneless organelles by liquid-liquid phase separation of intrinsically disordered proteins

• Autor(a) principal: Félix, Sara Sofia Gonçalves Sousa
• Data de Publicação: 2018
• Tipo de documento: Dissertação
• Idioma: eng
• Título da fonte: Repositório Científico de Acesso Aberto de Portugal (Repositórios Cientìficos)
• Texto Completo: http://hdl.handle.net/10362/56370
• topic: FUS; Liquid-liquid phase separation; membraneless organelles; intrinsically disordered proteins; intermolecular interactions; Domínio/Área Científica::Engenharia e Tecnologia::Engenharia Química

Subcellular compartmentalization allows the organization of complex biochemical reactions in space and time, underlying vital cell processes such as homeostasis, division and development. Several compartments, however, lack a physical barrier. These membraneless organelles are usually an assembly of stalled mRNA and proteins that undergo liquid-liquid phase separation (LLPS), being termed as ribonucleoprotein (RNP) granules. These proteinaceous liquids are usually involved in regulation of gene expression and nucleic acid processing. Proteins that drive LLPS process normally exhibit an overall unstructured composition, being referred as intrinsically disordered proteins (IDPs). Fused in sarcoma (FUS) is a ubiquitously expressed IDP, composed of several disordered domains such as the low complexity (LC) domain, three arginine-glycine-glycine (RGG) boxes and a proline-tyrosine nuclear localization signal (PY-NLS). In addition, FUS contains two globular domains involved in RNA-related functions, the RNA recognition motif (RRM) and the zinc finger (ZnF) domain. In certain stress conditions, FUS can undergo LLPS in the cytoplasm leading to formation of stress granules. Formation of these RNP granules increases the risk of self-templating protein fibrils that underpin fatal neurodegenerative diseases. Although the cell environment is known to play a crucial role in mediating RNP granule formation, the mechanisms and molecular determinants that drive LLPS are still unclear. In this context, the main objective of this work is to elucidate the influence of the environment on the formation of FUS granules and to comprehend the mechanisms behind LLPS process. For that purpose, it was explored the influence of the temperature, pH and abundant cellular metabolites, on the LLPS process. Using turbidity microplate assays it was possible to assess the degree of FUS LLPS under different conditions. FUS presented an upper critical temperature solution (UCST) phase separation, undergoing reversible phase separation at low temperature. It was observed that phase separation is significantly enhanced when FUS presents an overall neutral charge (pH 9.40) and in the presence of optimal concentration of charged metabolites, indicating that LLPS is mediated through electrostatic interactions. Moreover, stabilizing metabolites that induce protein compaction and the destabilization of hydrophobic interactions inhibited FUS LLPS process, suggesting that this process has also a hydrophobic character and that FUS structural disorder is crucial for FUS granule formation. Through NMR spectroscopy it was found that FUS globular domains undergo reversible cold denaturation at a temperature in which LLPS is enhanced. Together with the phase separation assays that demonstrated that RNA and Zn2+ enhance LLPS, it is proposed that both RRM and ZnF domain are involved in the phase separation process, undergoing electrostatic intermolecular interactions upon unfolding at low temperature. Remarkably, it was also showed that FUS maintains its overall structure upon phase separation. Using microscopic imaging, it was possible to observe FUS granules and to identify liquid-like characteristic such as wetting and fusion. Overall it was demonstrated that FUS LLPS process is extremely sensitive and controlled by the environmental conditions.