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; 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.

项目摘要 实现了基本的细胞功能，例如转录，RNA处理和DNA损伤修复 通过正确的空间和时间定位的生物分子络合物，其中包含数十个不同的生物分子复合物 蛋白质和核酸种。含有低复杂性氨基酸序列的RNA结合蛋白是 这些复合物的基本成分，但也形成神经退行性疾病的病理组件 和小儿癌。这些低复杂性序列在健康和疾病状态中的功能仍然 理解不佳，部分原因是很难获得高分辨率的结构信息 Teins参加了这些集会。 RNA结合蛋白EWING肉瘤断点1（EWSR1）是mem- 一组大约70种人类RNA结合蛋白的BER，这些蛋白包含本质上无序的低com- 带电氨基酸但含有高比例的芳族残基的丛性区域。 这些低复杂性区域自我关联，在通常的过程中驱动动态簇的组装 称为液态液相分离。 EWSR1主要在mRNA加工和成熟中发挥作用 通过形成动态，可逆的复合物，为脚手架提供脚手架并促进正确 处理机械的空间位置。低复杂区域的突变会导致不受控制的组件 - EWSR1（及其相关蛋白）的BLY形成与肌萎缩症进展相关的病理夹杂物 侧硬化，额叶痴呆症和相关神经病。此外，通过染色体易位 tions，EWSR1的低复合域连接到形成有效致癌融合的DNA结合域 负责小儿肉瘤的发展。关于分子结构信息很少 EWSR1的致病功能，尤其是低复杂性结构域如何有助于致癌 ewsr1 fusions的特性。 NMR光谱法的最新技术进步现在可以检测和 量化驱动复杂形成的动态，高度瞬态相互作用，从而提供了req- Uisite工具用于确定EWSR1的结构和功能。该项目将采用高级NMR规格 - TROSCOPIC和其他生物物理技术，荧光和流体动力学方法，光谱聚集 测定和显微镜与生化和生物学测定结合：（1）确定分子 导致EWSR1自组装和生物分子凝结的事件； （2）定义了如何 低复杂性域有助于正常和异常的EWSR1功能。 （3）确定 蛋白质形成和稳定的相位分离：涉及致癌EWS-FLI1的DNA复合物 融合蛋白。我们的调查结果将有助于促进我们对摩克隆 - 由低复合蛋白形成的动态蛋白/核蛋白复合物的ULAR组装。理解 定义其活动的结构性基础将指导制定治疗方法的策略 低复杂性蛋白或其分子伙伴。