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.

G蛋白偶联受体（GPCR）是一个大家族的膜蛋白， 启动细胞对广泛的细胞外信号的反应，如神经递质，激素或 光子因此，它们对许多生理过程至关重要，并且它们经常失调 导致人类疾病。这与FDA批准的>30%的药物靶向 GPCR。GPCR的主要作用机制是通过激活异源三聚体G蛋白， 开发高保真度和精确度监测G蛋白活性的工具对于阐明 许多神经递质，激素或药物的作用，并发现新的治疗剂。我们的目标是 开发一类新的遗传编码光学传感器，以监测异源三聚体的激活 G蛋白直接，在真实的时间和内源性水平的细胞。如果成功，我们的项目将提供 高精度G蛋白活性生物传感器，其（1）适于监测内源性G蛋白活性， 生理学相关的实验系统，（2）易于转移到其他研究者，（3）可扩展 以获得更高的吞吐量。因此，我们在这里提出开发的生物传感器将在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领域的发展至关重要，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的测量G蛋白的技术 迄今为止开发的激活仍然具有抑制进展的局限性。一个是他们依赖于异位 G蛋白的过度表达，从而损害读数的保真度。此外，要求 多个组分的同时过表达排除了它们在其中遗传学上 操纵并不容易。另一个可能的局限性是它们测量Gα-Gβγ亚基解离/ 重排，一种通过GPCR（其定义为 核苷酸交换）。 目的概要：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种G蛋白中每一种的GTP结合Gα亚基的形成来检测G蛋白活性 亚家族（Gi/o、Gs、Gq/11、G12/13）。我们的方法是基于生物传感器的模块化设计，分两步进行 过程在第一步（SA #1）中，我们将鉴定特异性结合到以下蛋白的活性Gα亚基的蛋白模块： 每个家族，并验证它们在并入双组分（供体/受体）时可以报告活性 BRET系统。在第二步（SA#2）中，生物传感器将适应于单分子系统，其报告 在天然表达的G蛋白的存在下，通过分子内BRET的活性。