Signal Transduction in the Primary Cilium: Hedgehog and Beyond

初级纤毛中的信号转导：刺猬及其他

• 批准号: 10457947
• 负责人: Benjamin Myers
• 金额: $ 38.13万
• 依托单位: UNIVERSITY OF UTAH
• 依托单位国家: 美国
• 财政年份: 2019
• 资助国家: 美国
• 起止时间: 2019-08-07 至 2024-07-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/10457947
• 关键词: Address; Affect; Animals; Area; Binding; Biochemical; Biochemistry; Biological; Biological Process; Biology; Biophysics; Cardiovascular system; Cell Communication; Cells; Cholesterol; Cilia; Congenital Abnormality; Development; Diagnostic; Disease; Embryonic Development; Ensure; Erinaceidae; Event; Functional disorder; Genes; Genetic; Genetic Diseases; Goals; Hair; Human Genetics; Human body; Ions; Link; Malignant Neoplasms; Membrane; Membrane Lipids; Metabolic; Modeling; Molecular; Organelles; Pathway interactions; Physiological; Process; Proteins; Research; Role; Sensory; Signal Pathway; Signal Transduction; Signaling Protein; Structure; Surface; System; Techniques; Testing; Therapeutic; Tissues; Work; animal tissue; body system; ciliopathy; experimental study; insight; meetings; organ growth; prevent; reconstitution; regenerative; sensor; smoothened signaling pathway; stem cell biology; tool; transcription factor

PROJECT SUMMARY / ABSTRACT: The primary cilium is a tiny, immotile antenna-shaped protrusion found on nearly every cell in the human body. This organelle is essential for organ development and function, serving as a hub for several critical cell signaling pathways. Perturbing cilia instigates a slew of ciliopathies that include developmental, sensory, metabolic, and regenerative disorders. While the biological roles of the cilium are known, it is unclear at a biochemical level how or why the cilium is needed for its constituent cascades to operate. This prevents an understanding of how the cilium serves any of its biological functions. It also stymies our ability to control ciliary signaling therapeutically. The goal of the work proposed here is to understand the biochemical and biophysical principles governing ciliary signal transduction. Rather than relying on conventional genetic and cell biological approaches in the field, we are harnessing non-traditional biochemical and physiologic tools from other areas of biology; this includes reconstitution systems and live-cell sensors for key transduction events. This proposal uses the Hedgehog (Hh) pathway, a model ciliary cascade and fundamental regulator of embryogenesis and stem cell biology, to address these outstanding questions. At the ciliary membrane, the seven-transmembrane (7TM) protein Smoothened (SMO) is the pivotal molecule controlling essentially all the Hh pathway’s biological activities. The following three projects will together uncover the molecules and mechanisms by which SMO and related ciliary 7TM proteins signal: 1) What are biochemical mechanisms that regulate SMO activation? This builds on the recent discovery that ion gradients and membrane lipids are key regulators of the crucial initial steps of Hh signaling. These studies now provide a platform to investigate how membrane cholesterol, a candidate “messenger” linking upstream pathway events to SMO activity, binds to and activates SMO. These efforts have also revealed additional SMO regulatory factors, which we will identify biochemically. 2) How does SMO, once activated, communicate to GLI transcription factors that control expression of Hh pathway target genes? We will pinpoint SMO’s long-sought immediate downstream effector in the Hh pathway and test the hypothesis that the cilium serves primarily as a “meeting place” to concentrate activated pathway components together. 3) How do these regulatory influences operate within the cilium? These experiments will test the physiological significance of the regulatory factors we identify biochemically, as well as quantitatively compare and selectively manipulate signal transduction in the ciliary and “cell body” compartments. Each project also provides stepping stones to investigate related mechanisms that govern 7TM proteins in many other ciliary signaling cascades. The techniques deployed and principles learned from the Hh cascade will thus be broadly applicable to other ciliary pathways. Defining these cascades at a mechanistic level is essential for the development of diagnostic and therapeutic tools for a range of devastating disorders; this includes congenital defects, malignancies, and dysfunction of the nervous, cardiovascular, and excretory systems.

项目摘要 /摘要：主要纤毛是发现的微小的，不易代的天线形状的蛋白 在人体几乎每个细胞上。该细胞器对于器官开发和功能至关重要，服务 作为几个关键细胞信号通路的枢纽。干扰纤毛刺激了一系列纤毛病，其中包括 发育，感觉，代谢和再生疾病。纤毛的生物学作用是 众所周知，在生化层面尚不清楚如何或为什么需要纤毛构成级联的操作。 这阻止了对纤毛如何服务其任何生物学功能的理解。这也阻碍了我们的能力 控制睫状信号传导理论。这里提出的工作的目的是了解生化 以及有关睫状信号转移的生物物理原理。而不是依靠常规遗传和 在田间的细胞生物学方法，我们正在利用非传统的生化和生理工具 生物学的其他领域；这包括用于关键转移事件的重建系统和实时传感器。 该提案使用刺猬（HH）途径，这是一种模型睫状级联和基本调节器 胚胎发生和干细胞生物学，以解决这些杰出的问题。在睫状膜上 七跨膜（7TM）蛋白平滑（SMO）是控制所有的关键分子 HH途径的生物学活动。以下三个项目将共同揭示分子和 SMO和相关睫状7TM蛋白信号的机制：1）什么是生化机制 调节SMO激活？这是基于最近的发现，即离子梯度和膜脂质是关键 HH信号传导关键初始步骤的调节器。这些研究现在提供了一个平台来调查如何 膜胆固醇是将上游途径事件与SMO活动联系起来的候选“ Messenger” 激活SMO。这些努力也揭示了其他SMO调节因素，我们将确定 生化。 2）SMO一旦激活，如何与控制的GLI转录因子进行交流 HH途径靶基因的表达？我们将确定SMO长期持久的下游效应器 在HH途径中，并检验了纤毛作为“聚会场所”的假设 激活的途径组件在一起。 3）这些监管影响如何在纤毛内运作？这些 实验将测试我们在生化上识别的调节因素的物理意义，以及 定量比较和选择性地操纵纤毛和“细胞体”室中的信号转导。 每个项目还提供了垫脚石来调查许多在许多人中控制7TM蛋白的机制 其他睫状信号级联。因此，部署的技术和从HH级联学到的原则将 广泛适用于其他睫状途径。将这些级联定义在机械水平上对于 开发用于一系列毁灭性疾病的诊断和治疗工具；这包括先天性 神经，心血管和正宗系统的缺陷，恶性肿瘤和功能障碍。