Protein dynamics underlying cilium-dependent Hedgehog signaling

纤毛依赖性 Hedgehog 信号传导的蛋白质动力学

基本信息

批准号：
10418376
负责人：
Radhika Subramanian
金额：
$ 33.6万
依托单位：
MASSACHUSETTS GENERAL HOSPITAL
依托单位国家：
美国
项目类别：
财政年份：
2022
资助国家：
美国
起止时间：
2022-09-20 至 2026-07-31
项目状态：
未结题

来源：
https://reporter.nih.gov/project-details/10418376
关键词：
Adult Basal cell carcinoma Binding Binding Proteins Binding Sites Biochemical Biological Biological Assay Biology Catalytic Domain Cell Nucleus Cell physiology Cell surface Cells Cilia Cilium Microtubule Complex Congenital Abnormality Consequentialism Craniofacial Abnormalities Cyclic AMP-Dependent Protein Kinases Data Distal Embryonic Development Erinaceidae GLI Family Protein Genetic Transcription Glioma Goals Hedgehog signaling complex location Homeostasis Human Image Impairment Importins In Vitro Kinesin Knowledge Light Link Location Malignant Neoplasms Mediating Microtubules Molecular Mutagenesis Mutation Newborn Infant Nuclear Nuclear Import Nuclear Localization Signal Nuclear Proteins Nuclear Translocation Oncogenes Organelles Output Pathway interactions Phosphotransferases Plus End of the Microtubule Positioning Attribute Process Property Protein Dynamics Proteins Protocols documentation Reaction Recombinant Proteins Regulation Research Resolution Series Signal Pathway Signal Transduction Signal Transduction Pathway Signaling Protein Site Structure Techniques Therapeutic Time Tissues Total Internal Reflection Fluorescent Transcription Coactivator Work base combat developmental disease experience gene repression hedgehog signal transduction imaging modality insight malformation medulloblastoma prevent programs protein complex protein protein interaction real-time images reconstitution recruit scaffold single molecule skeletal smoothened signaling pathway therapeutic development transcription factor tumor

项目摘要

ABSTRACT The Hedgehog (Hh) signaling pathway is a major intercellular signaling pathway important for embryonic development and adult tissue homeostasis. Errors in Hh signaling are linked to several newborn birth defects such as skeletal malformations and craniofacial defects, and associated with multiple tumors including basal cell carcinoma and medulloblastoma. An important but poorly understood aspect of vertebrate Hh signaling is the strict requirement of a microtubule-based organelle known as the primary cilium. While we now have a wealth of data on the “parts-list” of proteins involved in the Hh signaling, the molecular mechanisms underlying cilia-mediated signal transduction remains poorly understood. Our overall goal is to fill this major knowledge gap and provide a biochemical framework for the Hh signaling pathway by reconstituting key reaction of this pathway from its components. For this research, we will build on our experience in integrating single-molecule imaging methods with biochemical assays and cell biological readouts to connect the biochemical properties of the protein components to their cellular function. In this proposal, we focus a key step of the Hh signal transduction pathway which is the establishment of signaling complexes at the base and the tip of the cilia, as is needed for the proper activation or repression of the transcription factor Gli, the major effector of the Hh pathway. Here, we will: (1) define these protein-protein interactions through a series of reconstitution studies and determine how they restrict the Gli binding to the nuclear import machinery and (2) analyze the dynamics of key pathway proteins in the cilium using high-resolution real-time imaging. Together, these studies will define how Gli is regulated through dynamic transit between protein complexes at defined cytoplasmic locations. We expect that our findings will not only advance our understanding of the basic biology of this important signal transduction pathway but also shed light on how mutations in pathway components contribute to developmental disorders and human cancers.

抽象的 Hedgehog (Hh) 信号通路是对胚胎发育至关重要的主要细胞间信号通路 Hh 信号传导的错误与多种新生儿出生缺陷有关。例如骨骼畸形和颅面缺陷，并与包括基底细胞在内的多种肿瘤相关脊椎动物 Hh 信号传导的一个重要但知之甚少的方面是细胞癌和髓母细胞瘤。对称为初级纤毛的微管细胞器的严格要求。涉及 Hh 信号传导的蛋白质“部分列表”的大量数据，以及潜在的分子机制我们对纤毛介导的信号转导仍然知之甚少，我们的总体目标是填补这一主要知识。间隙并通过重建该信号通路的关键反应为 Hh 信号通路提供生化框架对于这项研究，我们将利用我们在整合单分子方面的经验。具有生化测定和细胞生物学读数的成像方法，以连接细胞的生化特性在本提案中，我们重点关注 Hh 信号的关键步骤。转导途径是在纤毛基部和尖端建立信号复合物，如转录因子 Gli（Hh 的主要效应子）的正确激活或抑制是必需的在这里，我们将：（1）通过一系列重构研究来定义这些蛋白质-蛋白质相互作用。并确定它们如何限制 Gli 与核输入机制的结合，并 (2) 分析动态这些研究将利用高分辨率实时成像来研究纤毛中的关键途径蛋白。定义 Gli 如何通过特定细胞质中蛋白质复合物之间的动态转运进行调节我们希望我们的发现不仅能增进我们对其基本生物学的理解。重要的信号转导途径，同时也揭示了途径成分的突变如何发挥作用发育障碍和人类癌症。