Steroid-Dependent Changes in the Yorkie Interactome

约克犬相互作用组中类固醇依赖性变化

• 批准号: 8688280
• 负责人: Kenneth H Moberg
• 金额: $ 26.99万
• 依托单位: EMORY UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2013
• 资助国家: 美国
• 起止时间: 2013-07-01 至 2017-04-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8688280
• 关键词: Address; Adhesions; Adhesives; Affect; Affinity Chromatography; Apoptosis; Apoptotic; Binding; Binding Proteins; Biochemical Genetics; Cadherins; Cell Adhesion Molecules; Cell Nucleus; Cell physiology; Cell-Cell Adhesion; Cells; Cessation of life; Complement; Complex; Contact Inhibition; Crowding; Cues; DNA Sequence Rearrangement; Data; Development; Disease; Drosophila genus; Drosophila melanogaster; EP300 gene; Ecdysone; Elements; Enhancers; Epithelial Cells; Epithelium; Estrogens; Event; Exposure to; Fatty acid glycerol esters; Gene Expression; Gene Targeting; Genetic; Goals; Growth; Hand; Heart; Histones; Homologous Gene; Hormones; Human; Hyperactive behavior; Immunoglobulins; Individual; Insecta; Integral Membrane Protein; Malignant Neoplasms; Mammals; Mass Spectrum Analysis; Mediating; Membrane; Mitotic; Modeling; Molecular Chaperones; Molecular Genetics; Mus; Mutation; Nuclear; Nuclear Protein; Nuclear Receptor Coactivator 3; Oncogene Proteins; Organ Size; Orthologous Gene; Pathway interactions; Physiological; Play; Proteins; Proteomics; Records; Recruitment Activity; Regulation; Reporter; Research Personnel; Role; Series; Signal Transduction; Staging; Steroid Receptors; Steroids; Techniques; Tissues; Transactivation; Transcription Coactivator; Transferase; Transgenic Organisms; Vertebrates; Whole Organism; Work; castration resistant prostate cancer; cell motility; ecdysone receptor; extracellular; fly; genetic analysis; imaginal disc; in vivo; malignant breast neoplasm; novel; public health relevance; receptor; research study; response; steroid hormone; tool; tumorigenesis; unpublished works

DESCRIPTION (provided by applicant): The Hippo (or alvador/Warts/Hippo, SWH) pathway is a well-conserved metazoan signaling cascade that restricts organ size in the fruit fly Drosophila melanogaster and limits tumorigenesis in mammals. The main target of this pathway is the Yorkie (Yki)/YAP1 transcriptional co-activator. Work from many labs studying Hippo and its target Yki/Yap1 in insect cells, human cells and mouse cells has coalesced around the hypothesis that the main regulators of this pathway are transmembrane molecules that mediate interactions between cells. According to this model, the main role of Yki/Yap1 is to serve as a key target of contact- inhibition mechanisms through which membrane-associated cell adhesion molecules 'sense' cell crowding and inhibit Yki/Yap1 to keep cells in a post-mitotic state. In our view this model is incomplete. As most epithelial cells spend their entire lives closely apposed with adjacent cells, large fluctuations in cell:cell adhesion seem to be an unlikely driver of the developmental growth that is normally dependent on Yki/Yap1. In unpublished work, we have found biochemical and genetic evidence of a novel form of adhesion-independent Yki regulation that plays a significant role in the physiologic growth of Drosophila tissues. We find that protein that mediate the cellular response to Ecdysone, the major steroid hormone in flies, are also required for cell:cell adhesion-independent modulation of Yki activity during normal, physiologic growth. Moreover, we can refine this Yki-regulatory effect to a molecular interaction between Yki itself and the Ec-responsive protein Taiman, whose human homolog Steroid Receptor Coactivator-3/Amplified-In-Breast Cancer-1 (SRC3/AIB-1) is amplified in a wide array of cancers. Our initial proteomic analysis of this Yorkie-Taiman complex has identified additional components that appear to define a macromolecular interaction network we have termed the Yorkie Nuclear Interactome (YNI). Our long-term goal is to understand how individual YNI proteins affect Yki-driven gene expression in Drosophila to identify mechanisms that trigger rearrangement of interactions within the YNI. The aim of the current studies is to use cutting-edge proteomic techniques to analyze the macromolecular composition of the YNI in the whole organism and in the presence of Ec, and to couple this with genetic analyses of the role of individual YNI components in Yki nuclear activity in developing imaginal discs. In the first aim o this proposal, we will use quantitative affinity purification-mass spectrometry (AP-MS) technique to carry out in vivo identification of the larger suite of YNI proteins in the physiologic setting f imaginal disc cells, with an additional focus on identification of proteins that are specifically recruited to the YNI or displaced from it following exposure to elevated levels of circulating Ec. In the second aim, we will use the diverse array of genetic tools in Drosophila to assess the role of individual YNI proteins (identified in the AP- MS experiments) in controlling Yki activity in developing tissues.

描述（由申请人提供）：Hippo（或 alvador/Warts/Hippo，SWH）途径是一种保守的后生动物信号级联，限制果蝇果蝇的器官大小并限制哺乳动物的肿瘤发生。该通路的主要靶标是 Yorkie (Yki)/YAP1 转录共激活因子。许多实验室研究 Hippo 及其在昆虫细胞、人类细胞和小鼠细胞中的靶标 Yki/Yap1 的工作都围绕这样一个假设：该途径的主要调节因子是介导细胞间相互作用的跨膜分子。根据该模型，Yki/Yap1 的主要作用是作为接触抑制机制的关键靶标，膜相关细胞粘附分子通过该机制“感知”细胞拥挤并抑制 Yki/Yap1 以保持细胞处于有丝分裂后状态。 在我们的 查看这个模型是不完整的。由于大多数上皮细胞一生都与相邻细胞紧密相连，因此细胞：细胞粘附的大幅波动似乎不太可能驱动通常依赖于 Yki/Yap1 的发育生长。在未发表的工作中，我们发现了一种新型的不依赖于粘附的 Yki 调节的生化和遗传证据，这种调节在果蝇组织的生理生长中发挥着重要作用。我们发现介导细胞对蜕皮激素（果蝇中主要类固醇激素）反应的蛋白质也是正常生理生长过程中 Yki 活性的细胞：细胞粘附独立调节所必需的。此外，我们可以将这种 Yki 调节效应细化为 Yki 本身和 Ec 响应蛋白 Taiman 之间的分子相互作用，Taiman 的人类同源类固醇受体辅激活因子-3/乳腺癌扩增-1 (SRC3/AIB-1) 在多种癌症中被扩增。我们对这种约克-泰曼复合物的初步蛋白质组学分析发现了其他成分，这些成分似乎定义了我们称之为约克核相互作用组（YNI）的大分子相互作用网络。我们的长期目标是了解单个 YNI 蛋白如何影响果蝇中 Yki 驱动的基因表达，以确定触发 YNI 内相互作用重排的机制。当前研究的目的是利用尖端蛋白质组学技术来分析整个生物体中以及在 Ec 存在的情况下 YNI 的大分子组成，并将其与单个 YNI 成分在 Yki 核活动中在发育成虫盘中的作用的遗传分析结合起来。 在本提案的第一个目标中，我们将使用定量亲和纯化-质谱（AP-MS）技术对成象盘细胞的生理环境中更大的 YNI 蛋白进行体内鉴定，并额外关注鉴定专门招募到 YNI 或在暴露于升高水平的循环 Ec 后从 YNI 中置换的蛋白质。在第二个目标中，我们将在果蝇中使用多种遗传工具来评估单个 YNI 蛋白（在 AP-MS 实验中鉴定）在控制发育组织中 Yki 活性方面​​的作用。