Understanding Patched1 protein and lipid interactions in cilia

项目总结/摘要 信号分子的时空调节对于发育和成体组织的稳态是至关重要的。的 Hedgehog途径在后生动物中是保守的，控制细胞增殖、分化， 迁移和干细胞维持。在脊椎动物中，Hedgehog信号通过初级纤毛传导 从大多数细胞的表面突出，包括癌症中的细胞，这些细胞是由 刺猬路径。肿瘤抑制因子Patched 1定位于纤毛并抑制Smoothened，一种刺猬 通路激活剂在通路激活后，Patched 1留下纤毛，并允许Smoothened激活 下游Hedgehog转录程序。Patched 1如何定位于纤毛并抑制Smoothened残留 未知该提议的中心假设是蛋白质相互作用的动态网络允许Patched 1 在纤毛中积累并调节纤毛脂质微环境以抑制Hedgehog信号转导。到 为了验证这一点，本提案的目的是确定纤毛蛋白的相互作用， Patched 1到纤毛，并确定Patched 1是否通过调节纤毛脂质来调节Smoothened 微环境 为了解决我们对Hedgehog信号理解的差距，我将利用最近的技术进步， 在蛋白质组邻近标记质谱、脂质组质谱和功能基因组学中， 我为这个提案设计的新模型系统。在目标1中，我将询问5种蛋白质相互作用物， Patched 1与人类疾病相关，可能是Patched 1在人类中积累和活性的基础。 初级纤毛在目标2中，我将定义Patched 1对睫状体脂质微环境的影响。结合起来，这些 目的是阐明Patched 1调节Hedgehog信号的生化机制。刺猬 通路误激活驱动髓母细胞瘤，最常见的儿科脑肿瘤，和基底细胞瘤。 癌症，美国最常见的癌症。因此，本提案将纳入与Hh相关的 癌细胞系，以确定作为Patched 1功能基础的睫状蛋白和脂质是否在癌细胞系中是保守的。 发展和疾病背景。总之，了解Patched 1，最经常发生突变的基因， 在Hh相关的癌症中，抑制Hedgehog信号传导将为人类生物学提供重要的见解， 可能为新疗法提供途径。

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.