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.

显着性：G蛋白偶联受体（GPCR）是一个大型膜蛋白家族， 对广泛的细胞外信号产生细胞反应，例如中性递质，激素或 照片。因此，它们对于许多物理过程至关重要，并且经常进行失调 导致人类疾病。这与30％的FDA批准药物目标相处得很好 GPCR。 GPCR作用的主要机理是通过激活异三聚体G蛋白的激活，因此 开发工具以高保真和精度监测G蛋白活性对于阐明的模式至关重要 许多神经递质，激素或药物的作用，并发现新颖的治疗剂。我们的目标是 开发一类新的遗传编码光学传感器来监测异三聚体的激活 G蛋白直接直接实时和在细胞中的内源性水平。如果成功，我们的项目将交付 高精度G蛋白活性生物传感器（1）可适应于监测内源G蛋白活性 与物理相关的实验系统，（2）容易转移到其他研究者，（3）可扩展 为了更高的吞吐量。这，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来研究G蛋白活性。 背景：荧光或生物发光共振能量转移的发展（FRET 或BRET）技术对于GPCR场的发展至关重要，允许敏感和精确 测量活细胞中G蛋白活性的测量。但是，测量G蛋白的基于RET的技术 迄今为止开发的激活仍具有达摩进展的局限性。一个是他们依靠生态 G蛋白的过表达，从而损害了读数的保真度。另外，要求 多个组件的简单过表达排除了它们在通用的系统中的使用 操纵并不容易。另一个可能的限制是它们测量Gα-Gβγ亚基解离/ 重排，近端但仍间接测量GPCR的G蛋白激活（由此定义 核苷酸交换）。 AIM的概要：我们设想了基于BRET的生物传感器的设计来监测内源性 G蛋白活性通过检测4 G蛋白中每种GTP结合Gα亚基的形成 亚家族（GI/O，GS，GQ/11，G12/13）。我们的方法基于两步的生物传感器的模块化设计 过程。在第一步（SA＃1）中，我们将确定特异性结合与活动Gα亚基的蛋白质模块 每个家庭并验证他们可以报告活动时可以报告活动（捐助者/受体） BRET系统。在第二步（SA＃2）中，生物传感器将适用于报告的单分子系统 分子内BRET的活性在固有表达的G蛋白的存在下。