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: 10665058
• 负责人: DAVID STEVEN LIBICH
• 金额: $ 31.9万
• 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/10665058
• 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; 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 基本的细胞功能，如转录，RNA加工和DNA损伤修复， 通过正确的空间和时间定位的生物分子复合物含有几十个不同的 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 这些复合物的基本成分，而且还形成神经退行性疾病中的病理组装体 和儿科癌症。这些低复杂性序列在健康和疾病状态中的功能仍然存在。 了解甚少，部分原因是难以获得高分辨率的结构信息的亲， 参加这些集会的团体。RNA结合蛋白尤因肉瘤断点1（EWSR 1）是一种蛋白质， 一组约70种人RNA结合蛋白，含有内在无序的低分子量的RNA， 复杂性区域缺乏带电氨基酸，但含有高比例的芳香族残基。 这些低复杂性区域自关联，通常在过程中驱动动态集群的组装 称为液-液相分离。EWSR 1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，提供支架，并促进正确的 加工机械的空间位置。低复杂性区域的突变导致不受控制的装配， EWSR 1（和相关蛋白）的bly形成与肌萎缩性疾病进展相关的病理性包涵体 侧索硬化、额颞痴呆和相关神经病。此外，通过染色体易位- EWSR 1的低复杂性结构域与DNA结合结构域结合，形成强致癌融合体 儿童肉瘤的形成目前还缺乏关于其分子结构的信息， EWSR 1的致病功能，特别是低复杂性结构域如何促进致癌基因的表达。 EWSR 1融合的特性。NMR光谱学中的最新技术进步现在使得能够检测和 量化驱动复杂形成的动态、高度瞬态相互作用，从而提供所需的 用于确定EWSR 1的结构和功能的可持续工具。该项目将采用先进的核磁共振技术， troscopic和其他生物物理技术，荧光和流体动力学方法，光谱聚集 分析和显微镜检查结合生物化学和生物学分析，以：（1）确定分子 导致EWSR 1自组装和生物分子缩合的事件;（2）定义如何 低复杂度结构域有助于正常和异常EWSR 1功能;和（3）确定的作用， 涉及致癌EWS-FLI 1的蛋白质：DNA复合物形成和稳定中的相分离 融合蛋白我们的调查结果将有助于我们推进对大分子的全面了解。 由低复杂性蛋白质形成的动态蛋白质/核蛋白复合物的有序组装。理解 定义其活性的结构基础将指导治疗靶向的策略的发展， 低复杂性蛋白质或其分子伴侣。