An Integrated Biochemical and Structural Approach to Delineating the Biology of EWSR1

描述 EWSR1 生物学的综合生化和结构方法

• 批准号: 10298663
• 负责人: DAVID STEVEN LIBICH
• 金额: $ 32.09万
• 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/10298663
• 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; 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; Investigation; Label; Light; Link; Liquid substance; Location; Macromolecular Complexes; Malignant Childhood Neoplasm; Measurement; Messenger RNA; Methods; MicroRNAs; Microsatellite Repeats; Microscopy; Mission; Molecular; Molecular Conformation; Molecular Structure; Monitor; Mutation; N-terminal; NMR Spectroscopy; Nature; Nerve Degeneration; Neurodegenerative Disorders; Neuropathy; Nucleic Acids; Nucleoproteins; Oncogenic; Pathogenicity; Pathologic; Phase; Physical condensation; Process; Property; Proteins; Public Health; RNA; RNA Processing; RNA Recognition Motif; RNA Splicing; RNA-Binding Proteins; Relaxation; Research; Resolution; Role; Signal Transduction; Specificity; Structure; Titrations; United States National Institutes of Health; Work; analytical ultracentrifugation; base; 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.

项目总结 实现了基本的细胞功能，如转录、RNA处理和DNA损伤修复 通过正确的时空定位包含数十种不同的生物分子复合体 蛋白质和核酸物种。包含低复杂性氨基酸序列的RNA结合蛋白包括 这些复合体的基本成分，但也形成神经退行性疾病的病理集合体 和儿科癌症。这些低复杂性序列在健康和疾病状态下的功能仍然存在 了解较少，部分原因是难以获得PRO-2的高分辨率结构信息。 参加这些集会的队员。RNA结合蛋白尤文肉瘤断裂点1(EWSR1)是mem-1。 一组大约70个人的RNA结合蛋白的BER含有固有的无序的低COM-... 缺乏带电氨基酸但含有高比例芳香族残基的复合体区域。 这些低复杂性区域自关联，通常驱动过程中动态集群的组装 称为液-液相分离。EWSR1在mRNA加工和成熟中的主要作用 通过形成动态的、可逆的复合体，为正确的 加工机械的空间位置。低复杂性区域的突变导致失控的Assem- EWSR1(及相关蛋白)形成与肌营养不良进展相关的病理性包涵体 侧索硬化症、额叶颞叶痴呆及相关神经病。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 负责儿童肉瘤的发展。目前尚缺乏分子结构信息。 EWSR1的致病功能，特别是低复杂性结构域在致癌中的作用 EWSR1-融合的性质。核磁共振光谱学的最新技术进步现在使检测和 量化驱动复杂地层的动态、高度瞬变的相互作用，从而提供要求- 用于确定EWSR1结构和功能的有用工具。该项目将采用先进的核磁共振技术- 透射镜和其他生物物理技术、荧光和流体动力学方法、光谱聚集 分析和显微镜结合生化和生物分析：(1)确定分子 导致EWSR1自组装和生物分子缩合的事件；(2)定义如何 低复杂性结构域有助于EWSR1的正常和异常功能；以及(3)决定 蛋白质形成和稳定过程中的相分离：涉及致癌EWS-FLI1的DNA复合体 融合蛋白。我们的研究结果将有助于增进我们对大分子乳胶的总体认识。 由低复杂性蛋白质形成的动态蛋白质/核蛋白复合体的有序组装。理解 确定其活动的结构基础将指导制定治疗靶向的策略 低复杂性蛋白质或其分子伴侣。