Imaging and manipulating oncoprotein phase separation and compartmentalization

成像和操作癌蛋白相分离和区室化

• 批准号: 10600830
• 负责人: Xiaokun Shu
• 金额: $ 42.13万
• 依托单位: UNIVERSITY OF CALIFORNIA, SAN FRANCISCO
• 依托单位国家: 美国
• 财政年份: 2021
• 资助国家: 美国
• 起止时间: 2021-04-01 至 2026-03-31
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/10600830
• 关键词: Address; Affect; Automobile Driving; Behavior; Binding; Biochemical; Biological; Biological Process; Biophysics; Cell Nucleus; Cells; Chemicals; Color; DNA Binding Domain; DNA Sequence; Development; Disease; Engineering; Fibroblasts; Gene Expression; Genetic; Genetic Transcription; Health; Human; Image; Label; Link; Liquid substance; MYCN gene; Malignant Childhood Neoplasm; Malignant Neoplasms; Mediator; Membrane; Modeling; Molecular; Neuroblastoma; Oncogenic; Oncoproteins; Pharmaceutical Preparations; Phase; Physical condensation; Play; Proteins; RNA Polymerase II; Rat-1; Reporting; Role; Signal Transduction; Structure; System; TRRAP gene; Technology; Tissues; Transcription Coactivator; Up-Regulation; Work; aurora kinase A; c-myc Genes; cell transformation; fluorescence imaging; high risk; insight; neuroblastoma cell; novel strategies; public health relevance; quantitative imaging; small molecule inhibitor; tool; transcription factor; transcriptome sequencing; tumor; tumorigenesis

Abstract MYC oncoproteins including c-Myc and MYCN are major drivers of human tumorigenesis. Expression of MYC is deregulated and enhanced in many types of human cancer. For instance, MYCN is a major driver of childhood cancers. While upregulated MYC expression induces tumor development in many tissues, depletion of MYC abolishes tumorigenesis and results in tumor regression in various tumor models. Despite significant progress in understanding the link between MYC upregulation and tumorigenesis, many key questions remain regarding how upregulated expression of MYC leads to tumorigenesis. The relationship between MYC upregulation and its transforming activity has not been investigated in a quantitative manner, which is important because one potential consequence of elevated expression is protein phase separation that occurs when protein concentration is above critical concentration. Recently, many transcription factors that contain intrinsically disordered region (IDR) have been reported to undergo concentration-dependent liquid-liquid phase separation (LLPS), forming biomolecular condensates (also known as membraneless compartments, liquid droplets). MYC oncoproteins are transcription factors and contain IDR and our preliminary study reveals punctate structures of MYCN in the nucleus of Kelly MYCN-amplified neuroblastoma cells, suggesting that MYCN forms condensates in the Kelly cells where MYCN is highly expressed. Functions of biological phase- separated condensates include compartmentalized signaling. Condensates of transcriptional factors have been proposed to compartmentalize transcriptional machineries and remodel gene transcription. MYC oncogenic signaling relies on its interaction with the key proteins including MAX and TRRAP for the transcriptional activity and transforming, and inhibition of these interactions results in MYC inactivation. To understand upregulated MYC oncoproteins and their link to tumorigenesis, several basic molecular biological questions need to be addressed regarding biophysical/chemical mechanisms and functional roles of MYC condensates, including: 1) Whether and how MYC undergoes phase separation and forms condensates in biochemical and cellular systems; 2) Whether MYC condensates compartmentalize transcriptional machineries; 3) Whether MYC phase separation remodels downstream gene expression and whether condensates of MYC is necessary for the transforming activity. Because LLPS is dependent on protein concentration, biological functions of phase separation are often entangled with effects from changes of expression levels. To dissect MYC LLPS and functional consequences, there is a need for chemogenetic tools that can manipulate MYC LLPS without changing protein levels, and ideally such manipulation can be performed in two opposite directions: driving condensate formation and dissolving existing condensates. Here we propose to develop such chemogenetic tools and combine them with fluorescent protein labeling technology and multicolor fluorescence imaging to dissect biochemical mechanisms and functional roles of MYCN condensates.

摘要 MYC癌蛋白包括c-Myc和MYCN，是人类肿瘤发生的主要驱动因素。MYC基因的表达 在许多类型的人类癌症中被解除管制和增强。例如，MYCN是一个主要的驱动力 儿童癌症。虽然MYC表达上调会在许多组织中诱导肿瘤发展，但枯竭 在不同的肿瘤模型中，MYC的作用是消除肿瘤的发生并导致肿瘤消退。尽管意义重大 了解MYC上调与肿瘤发生之间关系的进展，仍有许多关键问题 关于MYC的表达上调如何导致肿瘤的发生。MYC与MYC的关系 上调及其转化活性尚未得到定量研究，这一点很重要 因为表达升高的一个潜在后果是蛋白质相分离，当 蛋白质浓度高于临界浓度。最近，许多含有转录因子的 据报道，固有无序区(IDR)经历了浓度依赖的液-液 相分离(LLP)，形成生物分子冷凝物(也称为无膜室， 液滴)。MYC癌蛋白是转录因子，含有IDR，我们的初步研究揭示 Kelly MYCN扩增的神经母细胞瘤细胞核内MYCN的点状结构 MYCN在高表达MYCN的Kelly细胞中形成凝聚体。生物相的功能-- 分离的冷凝物包括分隔的信令。转录因子的缩合物已经被 建议划分转录机制并重塑基因转录。MYC致癌 信号依赖于它与包括MAX和TRRAP在内的关键蛋白的相互作用来实现转录活性 转化，抑制这些相互作用会导致MYC失活。理解被上调的 MYC癌蛋白及其与肿瘤发生的联系，几个基本的分子生物学问题需要 论述了MYC凝析油的生物物理/化学机制和功能作用，包括：1) MYC在生化和细胞中是否以及如何经历相分离和形成冷凝物 系统；2)MYC冷凝物是否使转录机制区隔；3)MYC阶段 分离重塑下游基因表达，以及MYC的凝集物是否对 转型活动。由于LLP依赖于蛋白质浓度，因此相的生物功能 分离往往与表达水平变化的影响纠缠在一起。要剖析MYC有限责任公司和 功能后果，需要化学发生工具来操纵MYC LLP，而不需要 改变蛋白质水平，理想情况下，这样的操作可以在两个相反的方向上进行：驾驶 凝析油的形成和溶解现有的凝析油。在这里，我们建议开发这种化学发生 工具，并将它们与荧光蛋白标记技术和多色荧光成像相结合，以 剖析MYCN凝析油的生化机制和功能作用。