Understanding Patched1 protein and lipid interactions in cilia

项目摘要/摘要 信号分子的时空调节对于发育和成人组织的动态平衡至关重要。这个 Hedgehog途径在后生动物中是保守的，控制着细胞的增殖、分化， 迁移和干细胞维持。在脊椎动物中，刺猬的信号是通过初级纤毛传递的 它从大多数细胞的表面投射出来，包括癌症中由失活的 刺猬小路。肿瘤抑制因子Patched1定位于纤毛并抑制刺猬Smoothened 途径激活剂。在通路激活时，Patched1离开纤毛，并允许Smoothed激活 刺猬下游转录程序。Patched1如何定位于纤毛并抑制光滑的残留 未知。这一提议的中心假设是，蛋白质相互作用的动态网络允许Patched1 在纤毛中蓄积，调节纤毛脂微环境，抑制刺猬信号转导。至 测试这一点，这一建议的目标是定义定位所需的睫状蛋白相互作用 Patched1到纤毛，并确定Patched1是否通过调节纤毛脂质来调节光滑 微环境。 为了解决我们对Hedgehog信号的理解上的差距，我将利用最近的技术进步 在蛋白质组邻近标记质谱仪、脂体质谱仪和功能基因组学中使用 我为这项提议生成了新的模型系统。在目标1中，我将询问5个蛋白质相互作用因子 Patched1与人类疾病有关，可能是Patched1积累和活性的基础 初级纤毛。在目标2中，我将定义Patched1对睫状脂微环境的影响。加在一起，这些 AIMS将阐明Patched1调节Hedgehog信号的生化机制。刺猬 通路失活导致髓母细胞瘤，最常见的儿童脑肿瘤，和基底细胞 癌症，美国最常见的癌症。因此，该提案将纳入与HH相关的 确定Patched1功能下的纤毛蛋白和脂类是否在 发展和疾病背景。总而言之，理解Patched1，最常见的突变基因是如何 在HH相关癌症中，抑制Hedgehog信号将为人类生物学和 潜在地为新的疗法提供了途径。

PROJECT SUMMARY/ABSTRACT Spatiotemporal regulation of signaling molecules is critical for development and adult tissue homeostasis. The Hedgehog pathway, which is conserved across metazoan animals, controls cell proliferation, differentiation, migration, and stem cell maintenance. In vertebrates, Hedgehog signals are transduced through primary cilia that project from the surface of most cells, including cells in cancers that are driven by misactivation of the Hedgehog pathway. The tumor suppressor Patched1 localizes to cilia and inhibits Smoothened, a Hedgehog pathway activator. Upon pathway activation, Patched1 leaves cilia and allows Smoothened to activate the downstream Hedgehog transcriptional program. How Patched1 localizes to cilia and inhibits Smoothened remain unknown. The central hypothesis of this proposal is that a dynamic network of protein interactions allow Patched1 to accumulate in cilia and regulate the ciliary lipid microenvironment to inhibit Hedgehog signal transduction. To test this, the objective of this proposal is to define the ciliary protein interactions necessary for localization of Patched1 to the cilia, and to determine if Patched1 regulates Smoothened by regulating the ciliary lipid microenvironment. To address the gaps in our understanding of Hedgehog signaling, I will leverage recent technical advances in proteomic proximity-labeling mass spectrometry, lipidomic mass spectrometry, and functional genomics using novel model systems I have generated for this proposal. In Aim 1, I will interrogate 5 protein interactors of Patched1 that are associated with human disease and may underlie Patched1 accumulation and activity in primary cilia. In Aim 2, I will define the impact of Patched1 on the ciliary lipid microenvironment. Combined, these aims will elucidate the biochemical mechanism by which Patched1 regulates Hedgehog signaling. Hedgehog pathway misactivation drives medulloblastoma, the most common pediatric brain tumor, and basal cell carcinoma, the most common cancer in the United States. Thus, this proposal will incorporate Hh-associated cancer cell lines to determine if ciliary proteins and lipids underlying Patched1 functions are conserved across developmental and disease contexts. In sum, understanding how Patched1, the most recurrently mutated gene in Hh-associated cancers, inhibits Hedgehog signaling will provide significant insights into human biology and potentially provide avenues for novel therapies.