Site-Specific Photochemistry on Epigenetic Readers for Interactome Profiling

抽象的。蛋白质中的赖氨酸乙酰化对细胞分化和 发展。在分子水平上，称为溴域的效应器模块识别 乙酰化赖氨酸通过结合大分子促进下游信号转导 特定基因组位点的复合体。尽管它们在生物学和疾病中扮演着基本的角色，但 缺乏对特定溴域的乙酰化相互作用伙伴的无偏见编目 可能是由于这种互动的弱、高度动态和依赖于上下文的性质。 然而，这样的分子信息对于描述精确的机制是必不可少的 溴结构域参与了复杂的转录调控过程，在生物学和 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和 通过在疏水性中引入光交联型氨基酸的启动子图谱(IBPP) 捕获瞬间相互作用伙伴的溴结构域笼子，随后是它们的蛋白质组 和基因组特征。我们将IBPP应用于BET溴域(BRD2、BRD3、BRD4 和BRDT)，以便从完整的细胞中识别它们的相互作用伙伴。后续生化 对新发现的相互作用组的验证和功能研究将导致改进 正常人乙酰化和溴域介导基因调控的机制研究 细胞以及人类恶性肿瘤中的细胞。我们方法的显著特点包括能够 识别具有高时间控制的弱结合伙伴和瞬时结合伙伴(由于交联性) (由于光作为干扰剂)BET家族密切相关成员(通过蛋白质) 具有非自然突变的工程)以特定细胞类型的方式。由于发生了交联化 在深埋在溴域的芳香笼子中，在鉴别正品时具有选择性 与染色质免疫沉淀相比，相互作用的伙伴预计要高得多。 (芯片)依赖于基于甲醛的非选择性交联法。成功 IBPP的含义将在提供分子水平和系统水平方面具有无价的价值 对60多种含溴结构域人体动态功能的认识 蛋白质。

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.