Novel Developmental Regulation of Bmp and nodal signaling by Tril

我们的长期目标是了解BMP和TGF-β/nodal信号在发育和疾病中的调节。 Bmp和TGF-β/nodal信号的不平衡导致出生缺陷和癌症，然而， 维持这些途径之间的平衡是不完全的。我们发现Tril是一种蛋白质， 调节BMP和节点信号，并提出它是通过一种新的机制，涉及Toll样 受体（Tlrs）。类铎受体在介导先天免疫反应中扮演进化保守的角色。拟议 研究将首次证明Tlr信号传导也是脊椎动物早期模式化所必需的， 并将为未来的研究奠定基础，研究Tril或下游信号成分是否发挥作用。 在先天性异常和疾病中的保护和/或因果作用。我们发表的研究表明Tril触发了 Smad 7的降解，以增强非洲爪蟾原肠胚形成过程中BMP信号，我们的新数据表明， Tril同时抑制节点信号。我们的数据支持这样的假设，即Tril作为一种共受体， Tlrs启动信号级联反应，在该信号级联反应中，PKK激酶激活Pellino 2。然后Pellino 2刺激Rnf 12- Smad 7介导的降解以增强Bmp信号传导，以及Nedd 4L介导的Nodal 信令组件以抑制节点信令。我们将测试这个假设如下（1）确定Tril 调节Nedd 4l以抑制节点信号传导。我们将比较结途径失活的动力学，分析 结受体的稳态水平，并询问Nedd 4l是否足以拯救控制中的结信号传导 和Tril缺陷的非洲爪蟾胚胎和小鼠胚胎成纤维细胞（MEF）。我们将测试Tril是否能阻止 蛋白体降解和/或刺激Nedd 4l的翻译后修饰。(2)确定是否以及 Tril如何调节Rnf 12介导Smad 7的降解。我们将测试Tril是否能够触发 Smad 7的降解在胚胎或其中Rnf 12的表达被敲低的MEFs中受损，并且 相反，Rnf 12介导的Smad 7降解是否在Tril morphants和tril突变体MEFs中受损。 我们将测试Rnf 1的稳态水平、亚细胞定位和/或翻译后修饰是否与细胞内的蛋白质水平有关。 在Tril变形胚胎和tril突变MEF中改变。(3)确定Tril是否激活TLR信号传导 级联调节BMP和节点信号传导。我们将下调Tlr 3、Tlr 4、pellino 2和pellino 3的功能， 在胚胎和MEFs中，并询问Bmp信号是否受损，节点信号是否增强，我们 将上调每种蛋白质的功能，并询问这是否能拯救Tril中的BMP和节点信号传导 morphant胚胎和tril突变MEFs。我们将测试Tril是否需要调节Pellino 2 在一些实施方案中，本发明的方法可以用于检测Smad 7和Nedd 4l的磷酸化、亚细胞定位和/或结合，并且将使用拯救测定来检测Smad 7和Nedd 4l的结合。 确定Pellino 2的磷酸化和/或其遍在蛋白连接酶活性是否是功能所需的 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.