Protein dynamics underlying cilium-dependent Hedgehog signaling

纤毛依赖性 Hedgehog 信号传导的蛋白质动力学

• 批准号: 10812097
• 负责人: Radhika Subramanian
• 金额: $ 20万
• 依托单位: MASSACHUSETTS GENERAL HOSPITAL
• 依托单位国家: 美国
• 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/10812097
• 关键词: Adult; Basal cell carcinoma; Binding; Biochemical; Biological; Biological Assay; Biology; Cell physiology; Cells; Cilia; Complex; Congenital Abnormality; Craniofacial Abnormalities; Cytoplasm; Data; Embryonic Development; Erinaceidae; Goals; Homeostasis; Human; Knowledge; Link; Location; Malignant Neoplasms; Mediating; Microtubules; Molecular; Mutation; Newborn Infant; Nuclear Import; Organelles; Pathway interactions; Property; Protein Dynamics; Proteins; Reaction; Research; Resolution; Series; Signal Pathway; Signal Transduction; Signal Transduction Pathway; Tissues; Work; base; combat; developmental disease; experience; gene repression; hedgehog signal transduction; imaging modality; malformation; medulloblastoma; protein complex; protein protein interaction; real-time images; reconstitution; single molecule; skeletal; smoothened signaling pathway; therapeutic development; transcription factor; tumor

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) 信号通路是细胞间信号通路的重要组成部分。 Hh 信号传导的错误与胚胎发育和成人组织稳态有关。 一些新生儿出生缺陷，例如骨骼畸形和颅面缺陷，以及 与多种肿瘤相关，包括基底细胞癌和髓母细胞瘤。 脊椎动物 Hh 信号传导的一个重要但知之甚少的方面是对 基于微管的细胞器被称为初级纤毛，而我们现在拥有丰富的数据。 在参与 Hh 信号传导的蛋白质的“部分列表”上，其分子机制 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个专业的空白。 知识差距并为 Hh 信号通路提供生化框架 在本研究中，我们将构建该途径的关键反应。 基于我们将单分子成像方法与生化分析相结合的经验 细胞生物学读数将蛋白质成分的生化特性与其 在本提案中，我们重点关注 Hh 信号转导途径的关键步骤。 这是在纤毛基部和尖端建立信号复合物，如 转录因子 Gli（主要效应子）的正确激活或抑制是必需的 在这里，我们将：（1）通过一系列的蛋白质-蛋白质相互作用来定义这些蛋白质之间的相互作用。 重构研究并确定它们如何限制 Gli 与核输入的结合 机器和（2）使用高通量分析纤毛中关键途径蛋白的动态 这些研究将共同​​确定 Gli 是如何通过调节的。 我们期望我们的蛋白质复合物在确定的细胞质位置之间进行动态转运。 研究结果不仅将增进我们对这一重要信号的基础生物学的理解 转导途径，还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。