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.

摘要。蛋白质中赖氨酸乙酰化对细胞分化和细胞分化有重要作用