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.

项目摘要 在肉瘤（FUS）中融合是一种核RNA结合蛋白，该蛋白经历了液态液相分离（LLP）。 当错误定位和/或失调时，FUS的异常相分离会导致致病的形成 与神经退行性疾病有关的固体样骨料，包括肌萎缩性侧索硬化症 （ALS）和额颞痴呆（FTD）。我们最近发现DDX6的降低，RNA 已知与FUS相互作用的解旋酶在应激期间显着减少细胞质FUS颗粒形成 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冷凝水 在体外，以浓度依赖性方式的数量和大小，即DDX6促进FUS凝结物的形成 在低浓度时，但限制了高于有限水平的生长，（ii）DDX6在FUS周围形成不连续的环 冷凝水。在这些令人兴奋的结果的基础上，我旨在测试DDX6调节FUS的假设 颗粒通过双重作用，作为颗粒核和皮克林剂。采摘剂是颗粒 吸附到冷凝水表面的不同特性，促进其流动性并保持较小 冷凝水大小。基于我们的结果，我们建议DDX6促进FUS颗粒成核，同时其作用 作为采摘剂，液滴的尺寸和流动性很小，从而防止了致病性聚集。 此外，我们预测ATP结合和RNA结构将影响DDX6对FUS颗粒的活性。我们 将在三个目标中测试这些预测。 AIM 1将利用体外冷凝测定法来评估相互作用 在DDX6，FUS，RNA和ATP之间，并确定DDX6是否充当皮克剂。 AIM 2将使用 生化和单分子测定法，以表征分子级的动力学 FUS，DDX6和RNA及其对ATP的依赖性。最后，AIM 3将使用基于细胞的方法调查 调节细胞内DDX6浓度和破坏ATP结合如何影响FUS颗粒的形成 野生型和与ALS相关的突变体。这项工作一起将导致对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。