Defining the Role of DDX6 in Regulating FUS Condensates

定义 DDX6 在调节 FUS 冷凝物中的作用

• 批准号: 10677605
• 负责人: Gemechu Mekonnen
• 金额: $ 4.77万
• 依托单位: JOHNS HOPKINS UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2022
• 资助国家: 美国
• 起止时间: 2022-08-01 至 2026-07-31
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/10677605
• 关键词: ATP Hydrolysis; ATP phosphohydrolase; Adsorption; Affect; Amyotrophic Lateral Sclerosis; Binding; Biochemical; Biological Assay; Biophysics; CRISPR/Cas technology; Caenorhabditis elegans; Cell Line; Cells; Cellular Stress; Complex; Cytoplasm; Cytoplasmic Granules; Defect; Dependence; Disease; Emulsions; Exhibits; Genetic Screening; Growth; Homeostasis; Human; In Vitro; Inclusion Bodies; Lead; Link; Liquid substance; Maintenance; Measures; Methods; Molecular; Mutation; Neurodegenerative Disorders; Nuclear RNA; PDAP2 Gene; Pathogenesis; Pathogenicity; Phase; Physical condensation; Play; Property; Proteins; RNA; RNA Helicase; RNA Probes; RNA-Binding Proteins; Regulation; Ribonucleoproteins; Role; Rotation; Route; Solid; Stimulus; Stress; Structure; Surface; Testing; Time; Work; cytotoxic; experience; experimental study; fluidity; frontotemporal lobar dementia amyotrophic lateral sclerosis; fused in sarcoma; helicase; knock-down; mutant; neuroblastoma cell; overexpression; particle; prevent; response; reverse genetics; single molecule; small hairpin RNA; stress granule; superresolution microscopy

PROJECT SUMMARY Fused in sarcoma (FUS) is a nuclear RNA binding protein that undergoes liquid-liquid phase separation (LLPS). When mislocalized and/or dysregulated, aberrant phase separation of FUS leads to the formation of pathogenic solid-like aggregates that are implicated in neurodegenerative diseases including amyotrophic lateral sclerosis (ALS) and frontotemporal dementia (FTD). We have recently discovered that reduction of DDX6, an RNA helicase known to interact with FUS, significantly diminishes cytoplasmic FUS granule formation during stress in neuroblastoma cells (SH-SY5Y). Our preliminary results show that (i) DDX6 modulates FUS condensate number and size in a concentration dependent manner in vitro i.e., DDX6 promotes FUS condensate formation at low concentrations but limits growth above a finite level and (ii) DDX6 forms a discontinuous ring around FUS condensates. Building on these exciting results, I aim to test the hypothesis that DDX6 regulates FUS granules through dual roles as a granule nucleator and Pickering agent. Pickering agents are particles with distinct properties which adsorb to the surface of condensates, promoting their liquidity and maintaining small condensate size. Based on our results, we propose that DDX6 promotes FUS granule nucleation while its role as a Pickering agent maintains small droplet size and liquidity, thus preventing pathogenic aggregation. Additionally, we predict that ATP binding and RNA structure will affect the activity of DDX6 on FUS granules. We will test these predictions in three aims. Aim 1 will utilize in vitro condensation assays to evaluate the interaction between DDX6, FUS, RNA and ATP and to establish whether DDX6 acts as a Pickering agent. Aim 2 will utilize biochemical and single molecule assays to characterize the molecular-level dynamics of the interaction between FUS, DDX6, and RNA, and its dependence on ATP. Finally, aim 3 will use cell-based methods to investigate how tuning intracellular DDX6 concentrations and disrupting ATP binding affects FUS granule formation in wildtype and ALS-associated mutants. Together, this work will lead to a deeper understanding of RNA-protein granule regulation which is of utmost importance to treating neurodegenerative diseases such as ALS and FTD.

项目概要 肉瘤融合蛋白 (FUS) 是一种经过液-液相分离 (LLPS) 的核 RNA 结合蛋白。 当定位错误和/​​或失调时，FUS 的异常相分离会导致致病性 与神经退行性疾病（包括肌萎缩侧索硬化症）有关的固体状聚集体 （ALS）和额颞叶痴呆（FTD）。我们最近发现 DDX6（一种 RNA）的还原 已知与 FUS 相互作用的解旋酶可显着减少应激期间细胞质 FUS 颗粒的形成 在神经母细胞瘤细胞（SH-SY5Y）中。我们的初步结果表明 (i) DDX6 调节 FUS 冷凝物 体外浓度依赖性方式的数量和大小，即 DDX6 促进 FUS 冷凝物形成 浓度较低，但限制生长超过有限水平，并且 (ii) DDX6 在 FUS 周围形成不连续的环 凝结物。基于这些令人兴奋的结果，我的目标是检验 DDX6 调节 FUS 的假设 通过作为颗粒成核剂和皮克林剂的双重作用来形成颗粒。 Pickering 剂是具有 吸附在冷凝物表面的独特特性，促进其流动性并保持较小的 凝结水尺寸。根据我们的结果，我们提出 DDX6 促进 FUS 颗粒成核，同时其作用 作为皮克林剂，可保持小液滴尺寸和流动性，从而防止病原体聚集。 此外，我们预测 ATP 结合和 RNA 结构将影响 FUS 颗粒上 DDX6 的活性。我们 将在三个目标上测试这些预测。目标 1 将利用体外缩合测定来评估相互作用 DDX6、FUS、RNA 和 ATP 之间的差异，并确定 DDX6 是否充当 Pickering 代理。目标 2 将利用 生化和单分子测定来表征之间相互作用的分子水平动态 FUS、DDX6 和 RNA 及其对 ATP 的依赖性。最后，目标 3 将使用基于细胞的方法来研究 调节细胞内 DDX6 浓度和破坏 ATP 结合如何影响 FUS 颗粒的形成 野生型和 ALS 相关突变体。总之，这项工作将带来对 RNA-蛋白质的更深入的了解 颗粒调节对于治疗 ALS 和 FTD 等神经退行性疾病至关重要。