An Integrated Biochemical and Structural Approach to Delineating the Biology of EWSR1 - Multi-Mode Detection and Imaging of Biomolecular Condensates.

描述 EWSR1 生物学的综合生化和结构方法 - 生物分子凝聚物的多模式检测和成像。

• 批准号: 10797858
• 负责人: DAVID STEVEN LIBICH
• 金额: $ 19.56万
• 依托单位: UNIVERSITY OF TEXAS HLTH SCIENCE CENTER
• 依托单位国家: 美国
• 财政年份: 2021
• 资助国家: 美国
• 起止时间: 2021-09-30 至 2026-06-30
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/10797858
• 关键词: Affinity; Amino Acid Sequence; Amino Acids; Amyotrophic Lateral Sclerosis; Automobile Driving; Behavior; Binding; Biochemical; Biological; Biological Assay; Biological Process; Biology; C-terminal; Cell model; Cell physiology; Cellular Assay; Charge; Chemicals; Childhood Leukemia; Chromosomal translocation; Code; Complex; Coupled; DNA; DNA Binding; DNA Binding Domain; DNA Repair; Detection; Development; Diffusion; Disease; EMSA; ETS Domain; EWS-FLI1 fusion protein; Environment; Event; Ewings sarcoma; Exhibits; FLI1 Transcription Factor; FLI1 gene; Family; Fluorescence Recovery After Photobleaching; Frontotemporal Dementia; Gene Expression; Genetic Transcription; Goals; Health; Human; Image; Investigation; Label; Link; Liquid substance; Location; Macromolecular Complexes; Malignant Childhood Neoplasm; Measurement; Messenger RNA; Methods; MicroRNAs; Microsatellite Repeats; Microscopy; Mission; Molecular; Molecular Conformation; Monitor; Mutation; N-terminal; NMR Spectroscopy; Nature; Nerve Degeneration; Neurodegenerative Disorders; Neuropathy; Nucleic Acids; Nucleoproteins; Oncogenic; Pathogenicity; Pathologic; Phase; Physical condensation; Process; Property; Protein Dynamics; Proteins; Public Health; RNA; RNA Processing; RNA Recognition Motif; RNA Splicing; RNA-Binding Protein EWS; RNA-Binding Proteins; Relaxation; Research; Resolution; Role; Signal Transduction; Specificity; Structure; TAF15 gene; Technology; Titrations; United States National Institutes of Health; Untranslated RNA; Work; analytical ultracentrifugation; biophysical techniques; childhood sarcoma; experimental study; in vivo; link protein; macromolecular assembly; member; mutant; neuron development; protein transport; recruit; reproductive development; scaffold; self assembly; stoichiometry; targeted treatment; therapeutic target; tool; tumorigenesis

PROJECT SUMMARY Fundamental cellular functions such as transcription, RNA processing, and DNA damage repair are achieved through the correct spatial and temporal localization of biomolecular complexes containing dozens of different protein and nucleic acid species. RNA-binding proteins that contain low-complexity amino acid sequences are essential components of these complexes but also form pathological assemblies in neurodegenerative diseases and pediatric cancers. The function of these low complexity sequences in healthy and disease states remain poorly understood, partly because of the difficulty in obtaining high resolution structural information of the pro- teins participating in these assemblies. The RNA binding protein Ewing sarcoma breakpoint 1 (EWSR1) is mem- ber of a group of approximately 70 human RNA-binding proteins that contain intrinsically disordered low-com- plexity regions that are deficient in charged amino acids but contain a high proportion of aromatic residues. These low complexity regions self-associate, driving the assembly of dynamic clusters in a process commonly referred to as liquid-liquid phase separation. EWSR1 primarily functions in mRNA processing and maturation through the formation of dynamic, reversible complexes that provide a scaffold for, and promote the correct spatial location of the processing machinery. Mutations in the low-complexity region cause uncontrolled assem- bly of EWSR1 (and related proteins) forming pathological inclusions linked to the progression of amyotrophic lateral sclerosis, frontal temporal dementia and related neuropathies. Further, through chromosomal transloca- tions, the low-complexity domain of EWSR1 is joined to DNA-binding domains forming potent oncogenic fusions responsible for the development of pediatric sarcomas. There is a paucity of molecular structural information on the pathogenic function of EWSR1 and particularly how the low-complexity domain contributes to the oncogenic properties of EWSR1-fusions. Recent technological advances in NMR spectroscopy now enable detection and quantification of the dynamic, highly transient interactions that drive complex formation, thus providing the req- uisite tool for determining the structure and function of EWSR1. This project will employ advanced NMR spec- troscopic and other biophysical techniques, fluorescent and hydrodynamic methods, spectroscopic aggregation assays and microscopy in conjunction with biochemical and biological assays to: (1) determine the molecular events leading to EWSR1 self-assembly and biomolecular condensation; (2) define the structural details of how the low complexity domain contributes to normal and abnormal EWSR1 functions; and (3) determine the role of phase separation in the formation and stabilization of protein:DNA complexes involving the oncogenic EWS-FLI1 fusion protein. The results of our investigations will help advance our general understanding about macromolec- ular assembly of dynamic protein/nucleoprotein complexes formed by low-complexity proteins. Understanding the structural basis that defines their activity will guide the development of strategies to therapeutically target low-complexity proteins or their molecular partners.

项目概要 实现转录、RNA 加工和 DNA 损伤修复等基本细胞功能 通过包含数十种不同生物分子复合物的正确时空定位 蛋白质和核酸种类。含有低复杂性氨基酸序列的 RNA 结合蛋白是 这些复合物的重要组成部分，但也形成神经退行性疾病的病理组合 和小儿癌症。这些低复杂性序列在健康和疾病状态下的功能仍然存在 理解甚少，部分原因是难以获得亲的高分辨率结构信息 参与这些集会的teins。 RNA 结合蛋白尤文肉瘤断点 1 (EWSR1) 是 mem- 一组大约 70 种人类 RNA 结合蛋白的 ber，这些蛋白含有本质上无序的低 com- 缺乏带电氨基酸但含有高比例芳香族残基的复杂区域。 这些低复杂性区域自关联，通常在一个过程中驱动动态簇的组装 称为液-液相分离。 EWSR1 主要在 mRNA 加工和成熟中发挥作用 通过形成动态的、可逆的复合物，为正确的反应提供支架并促进 加工机械的空间位置。低复杂性区域的突变会导致不受控制的组装 EWSR1（和相关蛋白）形成与肌萎缩性肌萎缩症进展相关的病理包涵体 侧索硬化症、额颞叶痴呆和相关神经病。此外，通过染色体易位 EWSR1 的低复杂性结构域与 DNA 结合结构域连接，形成有效的致癌融合 负责小儿肉瘤的发展。缺乏有关分子结构的信息 EWSR1 的致病功能，特别是低复杂性结构域如何促进致癌作用 EWSR1融合的特性。核磁共振波谱学的最新技术进步现在可以进行检测和 驱动复杂形成的动态、高度瞬态相互作用的量化，从而提供了要求 用于确定 EWSR1 结构和功能的 uisite 工具。该项目将采用先进的核磁共振技术 光学和其他生物物理技术、荧光和流体动力学方法、光谱聚合 化验和显微镜检查与生化和生物测定相结合，以：（1）确定分子 导致 EWSR1 自组装和生物分子缩合的事件； (2) 定义结构细节如何 低复杂度域有助于正常和异常的 EWSR1 功能； (3) 确定角色 蛋白质形成和稳定过程中的相分离：涉及致癌 EWS-FLI1 的 DNA 复合物 融合蛋白。我们的研究结果将有助于增进我们对大分子的一般理解 由低复杂性蛋白质形成的动态蛋白质/核蛋白复合物的线性组装。理解 定义其活性的结构基础将指导治疗目标策略的制定 低复杂性蛋白质或其分子伙伴。

项目成果

Biochemical and biophysical characterization of the nucleic acid binding properties of the RNA/DNA binding protein EWS.

RNA/DNA 结合蛋白 EWS 的核酸结合特性的生化和生物物理表征。

• DOI: 10.1002/bip.23536
• 发表时间: 2023
• 期刊: Biopolymers
• 影响因子: 2.9
• 作者: Selig,EmilyE;Bhura,Roohi;White,MatthewR;Akula,Shivani;Hoffman,ReneeD;Tovar,CarmelN;Xu,Xiaoping;Booth,RachellE;Libich,DavidS
• 通讯作者: Libich,DavidS

Insights into Molecular Diversity within the FET Family: Unraveling Phase Separation of the N-Terminal Low Complexity Domain from RNA-Binding Protein EWS.

深入了解 FET 家族内的分子多样性：从 RNA 结合蛋白 EWS 中解开 N 端低复杂性结构域的相分离。

• DOI: 10.1101/2023.10.27.564484
• 发表时间: 2023
• 期刊: bioRxiv : the preprint server for biology
• 作者: Johnson,CourtneyN;Sojitra,KandarpA;Sohn,ErichJ;Moreno-Romero,AlmaK;Baudin,Antoine;Xu,Xiaoping;Mittal,Jeetain;Libich,DavidS
• 通讯作者: Libich,DavidS