Site-Specific Photochemistry on Epigenetic Readers for Interactome Profiling

表观遗传读数器的位点特异性光化学用于相互作用组分析

• 批准号: 9289070
• 负责人: Kabirul Islam
• 金额: $ 29.11万
• 依托单位: UNIVERSITY OF PITTSBURGH AT PITTSBURGH
• 依托单位国家: 美国
• 财政年份: 2017
• 资助国家: 美国
• 起止时间: 2017-04-01 至 2022-02-28
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/9289070
• 关键词: Acetylation; Amber; Amino Acids; Autoimmune Diseases; BRD2 gene; Binding; Biochemical; Biological; Biological Markers; Biological Process; Biology; Bromodomain; Cataloging; Catalogs; Cell division; Cell physiology; Cells; Chromatin; Cognition Disorders; Collection; Crystallization; DNA; Development; Diagnosis; Disease; Disease model; Engineering; Environment; Epigenetic Process; Family; Formaldehyde; Gene Expression Regulation; Genetic Transcription; Genomics; Histones; Human; Hydrophobicity; Investigation; Laboratories; Length; Light; Link; Lysine; Macromolecular Complexes; Malignant Neoplasms; Mass Spectrum Analysis; Mediating; Methods; Modification; Molecular; Mutagenesis; Nature; Normal Cell; Photochemistry; Photosensitivity; Physiological; Point Mutation; Process; Protein Engineering; Proteins; Proteome; Proteomics; Reader; Reagent; Role; Signal Transduction; Site; Structure; System; Technology; Testing; Transcription Process; Transcriptional Regulation; Ultraviolet Rays; Validation; Work; base; cell type; chromatin immunoprecipitation; crosslink; drug discovery; genome-wide; human disease; improved; innovation; member; new therapeutic target; next generation sequencing; non-histone protein; novel; promoter; protein crosslink; public health relevance; spatiotemporal; tool; transcription factor

Abstract. Lysine acetylation in proteins contributes significantly to cellular differentiation and development. At the molecular level, effector modules called bromodomains recognize acetylated lysine to facilitate downstream signaling through binding of macromolecular complexes to specific genomic loci. Despite their fundamental role in biology and disease, an unbiased cataloguing of acetylated interacting partners of a specific bromodomain is lacking possibly due to the weak, highly dynamic and context-dependent nature of such interactions. However, such molecular information is essential in delineating precise mechanism by which bromodomains partake in the intricate process of transcriptional regulation in biology and disease. The current proposal outlines a unique strategy termed `Interaction-Based Protein and Promoter Profiling' (IBPP) by introducing a photo-crosslinkable amino acid into the hydrophobic cage of bromodomains to capture transiently interacting partners followed by their proteomic and genomic characterizations. We will apply IBPP to BET bromodomains (BRD2, BRD3, BRD4 and BRDT) to identify their interacting partners from intact cells. Subsequent biochemical validation and functional studies of the newly identified interactome will lead to improved mechanistic understanding of acetylation and bromodomain-mediated gene regulation in normal cells as well as in human malignancies. Salient points of our approach include the ability to identify weak and transient binding partners (because of crosslinking) with high temporal control (because of light as the perturbing agent) of closely related members of BET family (via protein engineering with unnatural mutagenesis) in cell-type specific manner. Since crosslinking occurs in the deeply embedded `aromatic cage' in bromodomains, selectivity in identifying authentic interacting partners is expected to be much higher compared to chromatin immunoprecipitation (ChIP) that relies on formaldehyde-based non-selective crosslinking method. Successful implication of IBPP will be invaluable in providing both molecular as well as system level understanding of the dynamic functions of more than sixty bromodomain-containing human proteins.

抽象。蛋白质中的赖氨酸乙酰化显著促进细胞分化， 发展在分子水平上，被称为布罗莫结构域的效应器模块识别 乙酰化赖氨酸，以通过结合大分子 与特定基因座的复合物。尽管它们在生物学和疾病中起着重要作用， 缺乏特异性溴结构域的乙酰化相互作用配偶体的无偏编目 这可能是由于这种相互作用的弱的、高度动态的和依赖于上下文的性质。 然而，这些分子信息对于描绘精确的机制是必不可少的， 布罗莫结构域参与生物学中转录调控的复杂过程， 疾病目前的提案概述了一种独特的策略，称为“基于相互作用的蛋白质”， 通过将可光交联的氨基酸引入到疏水性的“启动子分析”（IBPP）中， 溴结构域的笼，以捕获瞬时相互作用的伴侣，然后进行其蛋白质组学分析。 和基因组特征。我们将IBPP应用于BET溴结构域（BRD 2、BRD 3、BRD 4 和BRDT）以从完整细胞中鉴定它们的相互作用伴侣。后续生化 新鉴定的相互作用物组的验证和功能研究将导致改进的 乙酰化和溴结构域介导的基因调控机制的理解， 细胞以及人类恶性肿瘤。我们的方法的要点包括能够 鉴定具有高度时间控制的弱和瞬时结合配偶体（由于交联） （因为光作为扰动剂）BET家族的密切相关成员（通过蛋白质）的相互作用。 用非天然诱变进行工程化）。由于发生交联， 在溴结构域中深深嵌入的“芳香笼”中， 与染色质免疫沉淀相比， （ChIP），其依赖于基于甲酰胺的非选择性交联方法。成功 IBPP在提供分子水平和系统水平方面意义是非常宝贵的 了解60多个含溴结构域的人 proteins.