Understanding Patched1 protein and lipid interactions in cilia

了解纤毛中的 Patched1 蛋白和脂质相互作用

基本信息

批准号：
10688019
负责人：
Vikas daggubati
金额：
$ 5.27万
依托单位：
UNIVERSITY OF CALIFORNIA, SAN FRANCISCO
依托单位国家：
美国
项目类别：
财政年份：
2021
资助国家：
美国
起止时间：
2021-09-01 至 2024-08-31
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/10688019
关键词：
Ablation Address Adult Affinity Chromatography Animals Antibodies Automobile Driving Basal cell carcinoma Binding Biochemical Biological Assay Biological Models CRISPR interference Cancer cell line Cell Line Cell Maintenance Cell Proliferation Cells Childhood Brain Neoplasm Cilia Co-Immunoprecipitations Congenital Disorders Cryoelectron Microscopy Data Development Disease Erinaceidae Family Gene Expression Genes Genetic Genetic Transcription Goals Homeostasis Human Biology Immunofluorescence Immunologic Knowledge LLC-PK1 Cells Label Ligand Binding Lipid Binding Lipids Luciferases Malignant Neoplasms Malignant neoplasm of brain Mass Spectrum Analysis Mediating Membrane Transport Proteins Modeling Mutate Patch Tests Pathway interactions Patients Pediatric Neoplasm Peroxidases Proteins Proteomics Recurrence Regulation Reporter Signal Transduction Signaling Molecule Sterols Structure Surface System Testing Time Tissues Tumor Suppressor Proteins United States Vertebrates Western Blotting Work ascorbate base experimental study functional genomics hedgehog signal transduction human disease improved innovation insight lipidomics medulloblastoma medulloblastoma cell line migration novel novel therapeutics prevent programs smoothened signaling pathway spatiotemporal stem cells tool transcription factor

项目摘要

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.

项目概要/摘要信号分子的时空调节对于发育和成人组织稳态至关重要。这 Hedgehog 通路在后生动物中保守，控制细胞增殖、分化、迁移和干细胞维持。在脊椎动物中，Hedgehog 信号通过初级纤毛转导从大多数细胞的表面突出，包括由错误激活驱动的癌症细胞刺猬途径。肿瘤抑制因子 Patched1 定位于纤毛并抑制刺猬 Smoothened 途径激活剂。通路激活后，Patched1 离开纤毛并允许 Smoothened 激活下游 Hedgehog 转录程序。 Patched1 如何定位于纤毛并抑制 Smoothened 残留未知。该提案的中心假设是蛋白质相互作用的动态网络允许 Patched1 在纤毛中积累并调节纤毛脂质微环境以抑制 Hedgehog 信号转导。到对此进行测试，该提案的目的是定义定位所需的纤毛蛋白相互作用 Patched1 作用于纤毛，并确定 Patched1 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 信号传导理解上的差距，我将利用最新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使用我为这个提案生成了新颖的模型系统。在目标 1 中，我将询问 5 个蛋白质相互作用因子 Patched1 与人类疾病相关，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 对睫状脂质微环境的影响。综合起来，这些目标将阐明 Patched1 调节 Hedgehog 信号传导的生化机制。刺猬通路错误激活导致髓母细胞瘤（最常见的儿科脑肿瘤）和基底细胞癌，美国最常见的癌症。因此，该提案将纳入 Hh 相关的癌细胞系以确定 Patched1 功能背后的纤毛蛋白和脂质是否在跨细胞系中保守发育和疾病背景。总而言之，了解最常发生突变的基因 Patched1 是如何发生的在 Hh 相关癌症中，抑制 Hedgehog 信号传导将为人类生物学和可能为新疗法提供途径。