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: 10537448
• 负责人: Gemechu Mekonnen
• 金额: $ 4.68万
• 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/10537448
• 关键词: ATP Hydrolysis; ATP phosphohydrolase; Affect; Alpha Granule; Amyotrophic Lateral Sclerosis; Attenuated; 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; Microscopy; Molecular; Neurodegenerative Disorders; Nuclear RNA; PDAP2 Gene; Pathogenesis; Pathogenicity; Phase; Physical condensation; Play; Property; Proteins; RNA; RNA Helicase; RNA Probes; RNA-Binding Proteins; Regulation; Resolution; Ribonucleoproteins; Role; Rotation; Route; Seeds; Solid; Stimulus; Stress; Structure; Surface; Testing; Time; Work; base; 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

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 融合肉瘤（FUS）是一种核RNA结合蛋白，经历液-液相分离（LLPS）。 当错误定位和/或失调时，FUS的异常相分离导致致病性细胞因子的形成。 涉及神经变性疾病包括肌萎缩侧索硬化症的固体样聚集体 (ALS)额颞叶痴呆（FTD）我们最近发现DDX6的减少， 已知解旋酶与FUS相互作用，在胁迫期间显著减少细胞质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）DDX 6调制FUS凝聚 数量和大小在体外以浓度依赖性方式存在，DDX6促进FUS冷凝物形成 在低浓度下，但限制了有限水平以上的生长，以及（ii）DDX6在FUS周围形成不连续的环 冷凝物基于这些令人兴奋的结果，我的目标是测试DDX6调节FUS的假设 颗粒通过作为颗粒成核剂和Pickering剂的双重作用。Pickering试剂是颗粒， 吸附到冷凝物表面的独特性质，促进其流动性并保持小 凝析油尺寸。基于我们的研究结果，我们提出DDX 6促进FUS颗粒成核，而其作用 作为Pickering试剂，保持小液滴尺寸和流动性，从而防止致病性聚集。 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 将在三个目标中检验这些预测。目的1将利用体外缩合试验来评估相互作用 DDX6、FUS、RNA和ATP之间的关系，并确定DDX6是否充当Pickering剂。目标2将利用 生物化学和单分子测定，以表征分子水平的相互作用的动力学， FUS、DDX6和RNA，及其对ATP的依赖性。最后，aim3将使用基于细胞的方法来调查 调节细胞内DDX6浓度和破坏ATP结合如何影响FUS颗粒形成 野生型和ALS相关突变体。总之，这项工作将导致对RNA-蛋白质的更深入理解 颗粒调节，其对于治疗神经变性疾病如ALS和FTD是极其重要的。