Spatiotemporal visualization of adenylyl cyclase signaling

腺苷酸环化酶信号传导的时空可视化

• 批准号: 10664707
• 负责人: Makaia M. Papasergi
• 金额: $ 13.84万
• 依托单位: STANFORD UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2023
• 资助国家: 美国
• 起止时间: 2023-04-01 至 2025-03-31
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/10664707
• 关键词: A kinase anchoring protein; Address; Adenylate Cyclase; Architecture; Arrhythmia; Biochemical; Biological Assay; Biology; Cells; Cellular Morphology; Cellular biology; Chronic Obstructive Pulmonary Disease; Complex; Cryo-electron tomography; Cryoelectron Microscopy; Cyclic AMP; Cyclic AMP-Dependent Protein Kinases; Development; Diabetes Mellitus; Disease; Drug abuse; Environment; Enzymes; Fluorescence Microscopy; Funding; G Protein-Coupled Receptor Signaling; G-Protein-Coupled Receptors; GTP-Binding Protein alpha Subunits, Gs; GTP-Binding Proteins; Glaucoma; Goals; Heart Diseases; Human; Hyperthyroidism; In Situ; In Vitro; Label; Laboratories; Length; Lipids; Macromolecular Complexes; Malignant Neoplasms; Membrane; Mentors; Methodology; Methods; Migraine; Modeling; Molecular; Morphology; Multiple Partners; Outcome; Pathologic; Pathway interactions; Pharmacologic Substance; Physiological; Physiology; Positioning Attribute; Protein Biochemistry; Protein Isoforms; Protein Subunits; Proteins; Proteomics; Research; Research Personnel; Resources; Scaffolding Protein; Series; Signal Pathway; Signal Transduction; Structure; Therapeutic; Training; Tremor; Visualization; Work; beta-2 Adrenergic Receptors; differential expression; experience; flexibility; genetic regulatory protein; in vivo; nanodisk; neuropsychiatric disorder; novel; particle; phosphoric diester hydrolase; programs; protein purification; receptor; reconstitution; response; spatiotemporal; structural biology; therapeutic target

PROJECT SUMMARY/ABSTRACT Cyclic AMP (cAMP) is a critical secondary messenger that modulates many cell signaling pathways throughout physiology. Adenylyl cyclase (AC) catalytically converts ATP to cAMP in response to G protein- coupled receptor signaling, thus acting as an essential relay and integration center for cell signaling. Currently, ACs represent an unexploited target for treating a range of diseases including cancer, chronic obstructive pulmonary disease, neuropsychiatric disorders, diabetes, heart disease, and drug abuse. Within a cellular context, differential expression, compartmentalization, and microdomain localization of nine transmembrane AC isoforms give rise to a broad range of possible signaling outcomes, complicating our ability to therapeutically target this critical rheostat of cell signaling. Our mechanistic understanding of AC function is limited, with only a recent first glimpse of a full-length AC-Gαs structure revealed by cryo-electron microscopy (cryoEM). However, the static snapshot of AC in isolation forms an incomplete picture of the spatiotemporal signaling that occurs in vivo. The lack of molecular and mechanistic details of AC signaling assemblies in native environments leaves a gap in our understanding, further restricting the development of pharmaceuticals targeting AC and cAMP pathways. To address this gap, the overarching goal of this proposal is to capture physiologically relevant complexes between adenylyl cyclase and signaling partners in near-native and cellular environments. This will be achieved through the following specific aims: (1) structurally and biochemically characterize a functional signaling assembly of adenylyl cyclase, and (2) visualize adenylyl cyclase microdomain topography and its dynamics in response to cyclase activation in situ. Completing these aims will represent a substantial leap forward in adenylyl cyclase biology, providing the framework for further adenylyl cyclase study. This work builds upon my G protein biochemistry background and experience with cell signaling assays, protein purification, and fluorescence microscopy. Taking advantage of the world-class training and resources available in the laboratory of my mentor, Dr. Georgios Skiniotis, the proposed studies also provide an opportunity to acquire expertise in structural biology, cryo-electron tomography, proteomic approaches, sharpen my abilities as a researcher, and develop as an emerging leader in the adenylyl cyclase field. As a result, I will be well-positioned to establish a program of successfully funded independent research.

项目摘要/摘要 环状AMP（CAMP）是一个关键的次级信使，可调节许多细胞信号通路 整个生理学。腺苷酸环化酶（AC）将催化的ATP转换为cAMP，以响应G蛋白 耦合受体信号传导，因此充当细胞信号传导的必要继电器和整合中心。 目前，ACS代表了治疗一系列疾病的意外目标，包括癌症，慢性 阻塞性肺部疾病，神经精神疾病，糖尿病，心脏病和药物滥用。之内 细胞环境，差异表达，分隔和九个的微区域定位 跨膜AC同工型产生广泛的可能的信号传导结果，使我们的复杂 能够热靶向这种关键的细胞信号变阻器。我们对AC的机械理解 功能受到限制，最近仅首次瞥见了Cryo-Electron揭示的全长AC-GαS结构 显微镜（冷冻）。但是，孤立的交流的静态快照形成了不完整的图片 时空信号在体内发生。 AC信号传导缺乏分子和机械细节 本地环境中的集会在我们的理解中留下了差距，进一步限制了发展 针对交流和cAMP路径的药物。为了解决这一差距，这是总体目标 建议是捕获腺苷酸环化酶和信号伴侣之间的物理相关复合物 在近本和细胞环境中。这将通过以下特定目的实现：（1） 在结构和生化上表征了腺苷环酶的功能信号组件，（2） 可视化腺苷酸环化酶微域的地形及其对循环酶激活的动力学 原位。完成这些目标将代表adenylyl环酶生物学的实质性飞跃，提供 进一步的腺苷环酶研究的框架。这项工作以我的G蛋白生物化学为基础 背景和细胞信号测定，蛋白质纯化和荧光显微镜的经验。 利用我的导师博士实验室提供的世界一流培训和资源 Georgios Skiniotis，拟议的研究还为获得结构性专业知识提供了机会 生物学，冷冻电子层析成像，蛋白质组学方法，提高我作为研究人员的能力，并 发展成为腺苷循环酶领域的新兴领导者。结果，我将有充分的位置来建立 成功资助的独立研究的计划。