Novel Developmental Regulation of Bmp and nodal signaling by Tril

我们的长期目标是了解BMP和转化生长因子/结节信号在发育和疾病中是如何调节的。 BMP和转化生长因子/结节信号通路的不平衡会导致出生缺陷和癌症，但我们知道 维持这些途径之间的平衡是不完整的。我们鉴定了Tril是一种蛋白质，它能协调 调节BMP和节点信号，并提出它是通过一种涉及Toll样蛋白的新机制来实现这一点的 受体(TLR)。TLRs在调节先天免疫反应中扮演着进化上保守的角色。建议数 研究将首次证明TLR信号也是脊椎动物早期模式形成所必需的， 并将为未来的研究奠定基础，这些研究将询问TRIL或下游信令组件是否在 在先天性畸形和疾病中的保护性和/或因果作用。我们发表的研究表明，Tril触发了 Smad7的降解增强了非洲爪哇原肠发育过程中的BMP信号，我们的新数据表明 TRIL同时抑制结节信号转导。我们的数据支持这样的假设，即TRIL作为一种共同受体 TLRs启动IRAK激酶激活Pellino2的信号级联反应。然后Pellino2刺激RNF12- Smad7介导的降解增强BMP信号，以及Nedd4L介导的节点降解 抑制节点信令的信令组件。我们将对这一假设进行如下检验：(1)确定Tril如何 调节Nedd4l以抑制节点信号。我们将比较节点通路失活的动力学，分析 节点受体的稳态水平，并询问Ned4l是否足以挽救控制中的节点信号 和Tril缺陷的非洲爪哇胚胎和小鼠胚胎成纤维细胞(MEF)。我们将测试TRIL是否会阻止 蛋白酶体降解和/或刺激Nedd4l的翻译后修饰。(2)确定是否和 TRIL如何调节RNF12以调节Smad7的降解。我们将测试TRIL是否有能力触发 Smad7的降解在胚胎中或在Rnf12表达被下调的MEF中受损，并且 相反，Rnf12介导的Smad7的降解在TRIL变体和TRIL突变的MEF中是否受损。 我们将测试Rnf1的稳态水平、亚细胞定位和/或翻译后修饰是否 在TRIL变异胚胎和TRIL突变的MEF中发生改变。(3)确定TRIL是否激活TLR信令 级联来调节BMP和节点信号。下调TLR3、TLR4、IRAKS和Pellino2的功能 在胚胎和MEF中，询问BMP信号是否受损，结节信号是否增强，我们 我将上调每种蛋白质的功能，并询问这是否拯救了TRIL中的BMP和节点信号 变形胚胎和Tril突变的MEF。我们将测试TRIL是否需要监管Pellino2 磷酸化、亚细胞定位和/或与Smad7和Nedd4l结合，并将使用救援试验 确定是否需要Pellino2的磷酸化和/或其泛素连接酶活性来发挥作用 胚胎发生。

Our long term goal is to understand how Bmp and Tgf-ß/nodal signaling is regulated in development and disease. Imbalances in Bmp and Tgf-ß/nodal signaling lead to birth defects and cancers, and yet knowledge of how the equilibrium between these pathways is maintained is incomplete. We identified Tril as a protein that coordinately regulates Bmp and nodal signaling and propose that it does so through a novel mechanism involving Toll-like receptors (Tlrs). Tlrs play evolutionarily conserved roles in mediating innate immune responses. The proposed studies will provide the first demonstration that Tlr signaling is also required for early patterning in vertebrates, and will set the stage for future studies that ask whether Tril or downstream signaling components play a protective and/or causal role in congenital anomalies and disease. Our published studies show that Tril triggers degradation of Smad7 to enhance Bmp signaling during gastrulation in Xenopus, and our new data show that Tril simultaneously inhibits nodal signaling. Our data support the hypothesis that Tril serves as a co-receptor for Tlrs to initiate a signaling cascade in which Irak kinases activate Pellino2. Pellino2 then stimulates Rnf12- mediated degradation of Smad7 to enhance Bmp signaling, and Nedd4L-mediated degradation of nodal signaling components to inhibit nodal signaling. We will test this hypothesis as follows (1) Determine how Tril regulates Nedd4l to inhibit nodal signaling. We will compare the kinetics of nodal pathway inactivation, analyze steady state levels of nodal receptors and ask whether Nedd4l is sufficient to rescue nodal signaling in control and Tril deficient Xenopus embryos and mouse embryonic fibroblasts (MEFs). We will test whether Tril prevents proteosomal degradation and/or stimulates post-translational modification of Nedd4l. (2) Determine whether and how Tril regulates Rnf12 to mediate degradation of Smad7. We will test whether the ability of Tril to trigger degradation of Smad7 is impaired in embryos or in MEFs in which expression of Rnf12 is knocked down, and conversely, whether Rnf12-mediated degradation of Smad7 is impaired in Tril morphants and in tril mutant MEFs. We will test whether steady state levels, subcellular localization and/or post-translational modification of Rnf1 is altered in Tril morphant embryos and tril mutant MEFs. (3) Determine whether Tril activates a Tlr-signaling cascade to regulate Bmp and nodal signaling. We will down-regulate the function of Tlr3, Tlr4, Iraks and Pellino2 in embryos and in MEFs and ask whether Bmp signaling is impaired and nodal signaling is enhanced, and we will upregulate the function of each protein and ask whether this rescues Bmp and nodal signaling in Tril morphant embryos and tril mutant MEFs. We will test whether Tril is required to regulate Pellino2 phosphorylation, subcellular localization and/or binding to Smad7 and Nedd4l, and will use rescue assays to determine if phosphorylation of Pellino2 and/or its ubiquitin ligase activity are required for function during embryogenesis.