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: 9898385
• 负责人: Kabirul Islam
• 金额: $ 29.65万
• 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/9898385
• 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; chromatin remodeling; crosslink; drug discovery; genome-wide; genomic locus; 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.

抽象的。蛋白质中的赖氨酸乙酰化显着促进细胞分化和 发展。在分子水平上，称为溴结构域的效应器模块识别 乙酰化赖氨酸通过大分子结合促进下游信号传导 与特定基因组位点的复合物。尽管它们在生物学和疾病中发挥着重要作用， 缺乏对特定溴结构域的乙酰化相互作用伴侣的公正编目 可能是由于这种相互作用的弱性、高度动态性和环境依赖性。 然而，此类分子信息对于描绘精确的机制至关重要。 溴结构域参与生物学中复杂的转录调控过程 疾病。当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和 通过将可光交联的氨基酸引入疏水性分子中来进行启动子分析（IBPP） 布罗莫结构域笼捕获瞬时相互作用的伴侣，然后捕获其蛋白质组 和基因组特征。我们将 IBPP 应用于 BET 溴结构域（BRD2、BRD3、BRD4 和 BRDT）以从完整细胞中识别它们的相互作用伙伴。后续生化 新发现的相互作用组的验证和功能研究将导致改进 正常情况下乙酰化和溴结构域介导的基因调控的机制理解 细胞以及人类恶性肿瘤中。我们方法的要点包括能够 识别具有高时间控制的弱且短暂的结合伴侣（由于交联） （因为光作为扰动剂）BET家族密切相关的成员（通过蛋白质 以细胞类型特定的方式进行非自然诱变工程）。由于发生交联 在溴结构域中深深嵌入的“芳香笼”中，选择性地识别真实的 与染色质免疫沉淀相比，相互作用的伴侣预计要高得多 (ChIP) 依赖于基于甲醛的非选择性交联方法。成功的 IBPP 的含义对于提供分子水平和系统水平都具有不可估量的价值 了解 60 多个含溴结构域人类的动态功能 蛋白质。