Control of Notch signaling by endocytosis

描述(申请人提供)：Notch信号在所有后生动物的每个组织中都是普遍需要的，机制的缺陷与许多人类发育疾病和癌症有关。Notch途径的一个特殊特征是它的激活和调节在很大程度上依赖于内吞作用。Notch信号的一个神秘特征是，配体必须被信号细胞内化，才能激活相邻细胞上的Notch受体。我们提出的这项研究试图确定为什么配体内吞作用是信号传递所必需的。此外，我们还探讨了内吞因子的调节如何有助于信号的调节。两种内吞蛋白Epsin和Auxlin对于配体内化和信号转导是必不可少的。我们建议研究Epsin和Auxlin如何在信号细胞中发挥作用，以及它们是如何被调控的。特别是，将探索泛素化对Epsin活性的调节。此外，我们还建议确定信号细胞所需的其他内吞蛋白和调节因子。我们使用的方法包括果蝇遗传学、发育中眼睛的免疫组织化学和生物化学。首先，我们将确定Delta信号所需的Epsin的哪些蛋白质相互作用模块。我们将产生表达各种突变Epsin蛋白的转基因果蝇，并确定哪些突变支持信号传递、Delta内吞作用、Epsin泛素化、质膜定位和正常水平的Epsin积累。这些实验将解决有关Epsin的功能和调节的争议问题。其次，我们检验了两个假设，即为什么信号传递需要Auxlin。Auxlin在内吞作用中有不同的作用，如果我们能确定哪个作用对信号转导很重要，我们可能就能理解为什么Delta内吞作用是必要的。方法是测试不同转基因在果蝇中的表达是否会消除信号转导中对Auxlin的需求。此外，我们还对与生长素相互作用的基因进行筛选。第三，我们建议研究泛素调节Epsin的机制。Epsin被泛素化失活，而脂肪小面的去泛素化激活Epsin。我们的目标是了解这个泛素化循环与Delta信号的相关性。我们建议鉴定泛素化Epsin的泛素连接酶，分析缺乏连接酶的果蝇的表型，并在生化实验中使用该连接酶来确定Epsin泛素化的方式。此外，我们建议使用质谱学来绘制从苍蝇纯化的Epsin上泛素化的位置，并确定是否存在泛素链，如果存在，它们是如何连接的。第四，我们提出了遗传和生化实验来验证这一假设，即Ral GTP酶通过抑制Epsin的水平来负向调节Delta信号。第五，我们计划对我们在筛选中发现的九个与Epsin基因相互作用的基因进行表征，这些基因是液态的。我们认为，这些基因中的一些可能编码Delta的调节因子，通过Epsin来发挥作用。公共卫生相关性：Notch信号通路在后生动物中普遍用于控制细胞的增殖、规格、分化和每种细胞类型的生长。因此，Notch信号的缺陷与多种人类发育疾病和癌症有关。因此，了解Notch途径的调控是至关重要的，而结合果蝇遗传学和生物化学是研究这些过程的有力途径。

DESCRIPTION (provided by applicant): Notch signaling is required universally for development in every tissue in all metazoans, and defects in the mechanism are associated with many human developmental diseases and cancer. A peculiar feature of the Notch pathway is the extent to which its activation and regulation depend on endocytosis. A mysterious feature of Notch signaling is that ligand must be internalized by the signaling cells in order for them to activate the Notch receptor on adjacent cells. The research we propose seeks to determine why ligand endocytosis is necessary for signaling. In addition, we explore how regulation of endocytic factors contributes to regulation of signaling. Two endocytic proteins, Epsin and Auxilin, are absolutely necessary for ligand internalization and signaling. We propose to investigate how Epsin and Auxilin function in the signaling cells, and how they are regulated. In particular, regulation of Epsin activity by ubiquitination will be explored. Moreover, we propose to identify other endocytic proteins and regulators required in the signaling cells. The methodology we use includes Drosophila genetics, immunohistochemistry of developing eyes, and biochemistry. First, we will determine which protein interaction modules of Epsin are required for Delta signaling. We will generate transgenic flies that express a variety of mutant Epsin proteins, and determine which mutants support signaling, Delta endocytosis, Epsin ubiquitination, plasma membrane localization, and normal levels of Epsin accumulation. These experiments will resolve controversial issues regarding the function and regulation of Epsin. Second, we test two hypotheses as to why Auxilin is required for signaling. Auxilin has diverse roles in endocytosis, and if we can determine which role is important for signaling, we may be able to understand why Delta endocytosis is necessary. The approach is to test if expression of different transgenes in flies will obviate the requirement for Auxilin in signaling. In addition, we perform a screen for genes that interact with auxilin. Third, we propose to investigate the mechanism by which Ubiquitin regulates Epsin. Epsin is inactivated by ubiquitination, and deubiquitination by Fat facets activates Epsin. We aim to understand the relevance to Delta signaling of this ubiquitination cycle. We propose to identify the Ubiquitin-ligase that ubiquitinates Epsin, to analyze the phenotypes of flies that lack the ligase, and to use the ligase in biochemical experiments to determine the mode of Epsin ubiquitination. In addition, we propose to use mass spectrometry to map the sites of ubiquitination on Epsin purified from flies, and to determine whether Ubiquitin chains are present, and if so, how they are linked. Fourth, we propose genetic and biochemical experiments to test the hypothesis that the Ral GTPase negatively regulates Delta signaling by depressing the levels of Epsin. Fifth, we plan to characterize nine genes that we identified in a screen for genes that interact with the Epsin gene, liquid facets. We think that some of these genes are likely to encode regulators of Delta signaling that function through Epsin. PUBLIC HEALTH RELEVANCE: The Notch signaling pathway is used universally in metazoans to control cell proliferation, specification, differentiation, and growth of every cell type. Defects in Notch signaling are thus associated with a wide variety of human developmental diseases and cancer. For this reason, an understanding of Notch pathway regulation is critically important, and the combination of Drosophila genetics and biochemistry is a power manner in which to study these processes.