Next generation G protein activity biosensors

下一代 G 蛋白活性生物传感器

• 批准号: 9789949
• 负责人: Mikel Garcia-Marcos
• 金额: $ 20.63万
• 依托单位: BOSTON UNIVERSITY MEDICAL CAMPUS
• 依托单位国家: 美国
• 财政年份: 2018
• 资助国家: 美国
• 起止时间: 2018-09-22 至 2021-08-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/9789949
• 关键词: Adopted; Agreement; Binding; Biological Assay; Bioluminescence; Biosensor; Cell Surface Receptors; Cells; Data; Detection; Development; Dissociation; Drug Targeting; Energy Transfer; Engineering; Equipment; Etiology; Event; FDA approved; Family; Fluorescence; Fluorescence Resonance Energy Transfer; G Protein-Coupled Receptor Signaling; G-Protein-Coupled Receptors; GTP Binding; GTP Binding Domain; GTP-Binding Proteins; Genetic; Goals; Guanine Nucleotide Exchange Factors; Guanosine Triphosphate; Heterotrimeric GTP-Binding Proteins; Hormones; Image; Individual; Kinetics; Ligands; Luciferases; Measurement; Measures; Membrane Proteins; Mental Health; Molecular; Molecular Conformation; Monitor; Neurotransmitters; Nucleotides; Pathway interactions; Peptides; Pharmaceutical Preparations; Pharmacology; Photons; Physiological; Physiological Processes; Positioning Attribute; Process; Property; Protein Family; Proteins; Proxy; Reporting; Research Personnel; Resolution; Rest; Second Messenger Systems; Shrimp; Signal Transduction; Space Perception; Specificity; System; Techniques; Technology; Tertiary Protein Structure; Therapeutic Agents; Time; alpha helix; base; deep ocean; design; extracellular; genetic manipulation; human disease; improved; instrumentation; nanoluciferase; neuropsychiatric disorder; next generation; novel therapeutics; optical sensor; overexpression; polypeptide; protein activation; receptor; response; sensor; tool

SIGNIFICANCE: G protein-coupled receptors (GPCRs) are a large family of membrane proteins that initiate cellular responses to a wide range of extracellular signals, like neutrotransmitters, hormones or photons. Consequently, they are critical for many physiological processes and their dysregulation frequently leads to human disease. This is in good agreement with the fact that >30% of FDA-approved drugs target GPCRs. The main mechanism of action of GPCRs is through the activation of heterotrimeric G proteins, so developing tools to monitor G protein activity with high fidelity and precision is crucial to elucidate the mode of action of many neurotransmitters, hormones or drugs, and to discover novel therapeutic agents. Our goal is to develop a new class of genetically-encoded optical sensors to monitor the activation of heterotrimeric G proteins directly, in real time and at the endogenous level in cells. If successful, our project will deliver high precision G protein activity biosensors that are (1) adaptable to monitor endogenous G protein activity in physiologically relevant experimental systems, (2) readily transferable to other investigators, and (3) scalable for higher throughput. Thus, the biosensors we propose to develop here will have a profound impact in the field by enabling the study of G protein activity under native conditions with unprecedented detail and accuracy. BACKGROUND: The development of fluorescence or bioluminescence resonance energy transfer (FRET or BRET) techniques has been crucial for the advance of the GPCR field by allowing the sensitive and precise measurement of G protein activity in living cells. However, RET-based techniques to measure G protein activation developed to date still have limitations that dampen progress. One is that they rely on the ectopic overexpression of G proteins, thereby compromising the fidelity of readouts. Also, the requirement for simultaneous overexpression of multiple components precludes their use in systems in which genetic manipulation is not easy. Another possible limitation is that they measure Gα-Gβγ subunit dissociation/ rearrangement, a proximal but still indirect measure of G protein activation by GPCRs (which is defined by nucleotide exchange). SYNOPSIS OF AIMS: We have envisioned the design of BRET-based biosensors to monitor endogenous G protein activity by detecting the formation of GTP-bound Gα subunits for each one of the 4 G protein subfamilies (Gi/o, Gs, Gq/11, G12/13). Our approach is based on the modular design of biosensors in a two-step process. In the first step (SA#1), we will identify protein modules that specifically bind to active Gα subunits of each family and validate that they can report activity when incorporated into a two-component (donor/acceptor) BRET system. In the second step (SA#2), the biosensors will be adapted to a unimolecular system that reports activity by intramolecular BRET in the presence of natively expressed G proteins.

意义：G 蛋白偶联受体 (GPCR) 是一个膜蛋白大家族， 启动细胞对多种细胞外信号的反应，如中性递质、激素或 经过检查，它们对许多生理过程至关重要，而且它们经常失调。 这与 FDA 批准的超过 30% 的药物靶向这一事实非常吻合。 GPCR 的主要作用机制是通过异源三聚体 G 蛋白的激活，因此 开发高保真度和精确度监测 G 蛋白活性的工具对于阐明 G 蛋白活性的模式至关重要 我们的目标是研究许多神经递质、激素或药物的作用，并发现新的治疗药物。 开发一类新型基因编码光学传感器来监测异三聚体的激活 如果成功，我们的项目将直接在细胞内实时提供 G 蛋白。 高精度 G 蛋白活性生物传感器 (1) 适用于监测内源性 G 蛋白活性 生理相关的实验系统，（2）易于转移给其他研究人员，（3）可扩展 因此，我们建议在这里开发的生物传感器将对该领域产生深远的影响。 通过在天然条件下以前所未有的细节和准确性研究 G 蛋白活性。 背景：荧光或生物发光共振能量转移（FRET）的发展 或 BRET）技术对于 GPCR 领域的进步至关重要，因为它允许灵敏和精确的 活细胞中 G 蛋白活性的测量 然而，基于 RET 的技术可以测量 G 蛋白。 迄今为止开发的激活仍然存在阻碍进展的局限性，其中之一是它们依赖于异位。 G 蛋白的过度表达，从而损害读数的保真度。 多种成分的同时过度表达妨碍了它们在遗传性系统中的使用 另一个可能的限制是它们测量 Gα-Gβγ 亚基解离/。 重排，GPCRs 对 G 蛋白激活的近端但仍然间接的测量（其定义为 核苷酸交换）。 目标概要：我们设想设计基于 BRET 的生物传感器来监测内源性 通过检测 4 个 G 蛋白中每一个的 GTP 结合 Gα 亚基的形成来检测 G 蛋白活性 我们的方法基于两步生物传感器的模块化设计。 在第一步 (SA#1) 中，我们将鉴定与活性 G​​α 亚基特异性结合的蛋白质模块。 每个家庭并验证他们在纳入两个组成部分（捐赠者/接受者）时是否可以报告活动 在第二步（SA#2）中，生物传感器将适应报告的单分子系统。 在天然表达的 G 蛋白存在的情况下通过分子内 BRET 检测活性。