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.

项目总结/摘要 环磷酸腺苷（cAMP）是一种重要的第二信使，调节许多细胞信号通路 在整个生理学中。腺苷酸环化酶（AC）催化ATP转化为cAMP响应G蛋白- 偶联受体信号传导，因此充当细胞信号传导的必要中继和整合中心。 目前，AC代表了用于治疗一系列疾病的未开发的靶标，包括癌症、慢性炎症、糖尿病和其他疾病。 阻塞性肺病、神经精神疾病、糖尿病、心脏病和药物滥用。内 9个基因的细胞背景、差异表达、区室化和微区定位 跨膜AC亚型引起广泛的可能信号传导结果，使我们的研究复杂化。 能够治疗性地靶向细胞信号传导的关键变阻器。我们对AC的机械理解 功能是有限的，只有最近的第一次瞥见全长AC-Gαs结构揭示了冷冻电子 显微镜（cryoEM）。然而，孤立的AC的静态快照形成了对AC的不完整的描述。 发生在体内的时空信号。缺乏AC信号传导的分子和机制细节 本机环境中的程序集在我们的理解中留下了空白，进一步限制了开发 针对AC和cAMP通路的药物。为了弥补这一差距， 一个提议是捕获腺苷酸环化酶和信号伴侣之间的生理相关复合物 在近原生和蜂窝环境中。这将通过以下具体目标实现：（1） 从结构和生物化学上表征腺苷酸环化酶功能性信号传导组装，和（2） 可视化腺苷酸环化酶微区形貌及其对环化酶激活的响应动力学， 原地。完成这些目标将代表腺苷酸环化酶生物学的重大飞跃， 为腺苷酸环化酶的进一步研究提供了框架。这项工作建立在我的G蛋白生物化学基础上 细胞信号分析、蛋白质纯化和荧光显微镜的背景和经验。 利用我的导师，博士的实验室提供的世界一流的培训和资源。 Georgios Skiniotis，拟议的研究还提供了一个机会，获得结构方面的专业知识， 生物学，冷冻电子断层扫描，蛋白质组学方法，提高我作为研究人员的能力， 发展成为腺苷酸环化酶领域的新兴领导者。因此，我将很好地建立 一个成功资助的独立研究项目。