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。蛋白质中赖氨酸乙酰化对细胞分化和 发展。在分子水平上，效应子模块称为溴结构域识别 乙酰化赖氨酸通过大分子的结合来促进下游信号传导 特定基因组基因局的复合物。尽管它们在生物学和疾病中的基本作用，但 缺少特定溴ab域的乙酰化相互作用伙伴的无偏分组 可能是由于这种相互作用的弱，高度动态和上下文依赖性的性质。 但是，这种分子信息对于描述精确机制至关重要 溴化域参与生物学转录调节的复杂过程， 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 通过将光合链接氨基酸引入疏水性的启动子分析（IBPP） 溴结构域的笼子以捕获短暂相互作用的伴侣，然后是其蛋白质组学 和基因组特征。我们将应用IBPP赌注溴化域（BRD2，BRD3，BRD4 和BRDT）以确定其相互作用的伴侣从完整的细胞中。随后的生化 新确定的Interactome的验证和功能研究将导致改进 乙酰化和溴结构域介导的基因调节的机械理解 细胞以及人类恶性肿瘤。我们方法的重要点包括 确定具有高时间控制的弱和瞬态结合伙伴（由于交联） （由于光作为扰动剂）与BET家族密切相关的成员（通过蛋白质 具有不自然诱变的工程）以细胞类型的特异性方式进行。由于交联发生 在溴化群中深层嵌入的“芳族笼”中，选择性识别真实的选择性 与染色质免疫沉淀相比，相互作用的伴侣预计将高得多 （芯片）依赖于甲醛的非选择交联方法。成功的 IBPP的含义对于提供分子和系统级别将是无价的 理解六十多个含溴化域的人的动态功能 蛋白质。