在体外发光测试中可访问和通用,用于检测GPCR激活
Ruby M Miller1,2, Jennifer Sescil1,2, Marina C Sarcinella2
1Life Sciences Institute, University of Michigan, Ann Arbor, Michigan 48109, United States.
ACS measurement science au
|October 23, 2023
概括
一种新的体外方法,IGNiTR,使用分裂的纳米化酶来检测激活的G蛋白结合受体 (GPCR) 和它们的连接体. 这种多功能工具可以进行GPCR查和临床诊断.
科学领域:
- 生物化学 生物化学
- 分子生物学分子生物学
- 药理学 药理学是指药理学的学科.
背景情况:
- G蛋白结合受体 (GPCR) 对生理过程至关重要,是主要的药物标类.
- 现有的研究GPCR的方法通常需要复杂的基于细胞的测试或纯化的系统.
- 需要强大的,可适应的体外工具来表征GPCR-连接物相互作用.
研究的目的:
- 开发一种可通用的,基于细胞解质的体外方法来检测激活的GPCRs.
- 创建一个名为IGNiTR (体外GPCR分裂NanoLuc干触发报告器) 的工具,用于GPCR和干表征.
- 为了证明IGNiTR在不同GPCR类型和粘合剂格式中的广泛适用性.
主要方法:
- 设计了一个分裂的NanoLuc记者系统,在GPCR激活后被重组.
- 使用特定形状的结合剂 (纳米体,miniG蛋白质,模仿剂) 来弥合激活的GPCRs并分裂NanoLuc片段.
- 通过使用各种Gs-和Gi-合的GPCRs在细胞解质格式中验证了IGNiTR试验.
主要成果:
- 通过使用IGNiTR在多个GPCR和结合剂类型中成功复制了NanoLuc活性.
- 使用多巴胺受体D1.1进行GPCR配体查的概念证明.
- 展示了潜在的护理点应用的阿片类药物检测和纳米磁盘中的GPCR表征.
结论:
- IGNiTR为GPCR研究提供了一个多功能和可扩展的体外平台.
- 基于细胞溶酸盐的格式为连接体检测和查提供了方便和广泛的应用.
- IGNiTR促进了GPCR的表征,并有可能用于诊断应用.
相关概念视频
01:21G-protein Coupled Receptors
G-protein coupled receptors are ligand binding receptors that indirectly affect changes in the cell. The actual receptor is a single polypeptide that transverses the cell membrane seven times creating intracellular and extracellular loops. The extracellular loops create a ligand specific pocket which binds to neurotransmitters or hormones. The intracellular loops holds onto the G-protein.
02:11Reporter Genes
Reporter genes are a type of protein-coding gene that are often tagged to a gene of interest. Once inside a target cell, reporter genes usually produce visually identifiable characteristics like fluorescence and luminescence when expressed along with the gene of interest. Thus, reporter genes “report” the presence or absence of genes of interest in an organism, determine the gene expression pattern, or track the physical location of a DNA segment or protein in the cell.
Commonly used reporter...
Commonly used reporter...
01:21G-Protein Gated Ion Channels
GPCRs are primarily responsible for our sense of smell, taste, and vision. The binding of a sensory stimulus activates GPCR to stimulate effector proteins, many of which are ion channels in the sensory organs. GPCRs modulate the opening and closing of the target ion channels either directly by binding them, or by releasing second messengers that activate these channels. As ions move across the membrane, the membrane potential is altered, which induces an appropriate response.
Sensory organs,...
Sensory organs,...
01:30Transducer Mechanism: G Protein–Coupled Receptors
G Protein–Coupled Receptors (GPCRs) are membrane-bound receptors that transiently associate with heterotrimeric G proteins and induce an appropriate response to various stimuli. GPCRs regulate critical physiological pathways and are excellent drug targets for treating diseases such as diabetes, cancer, obesity, depression, or Alzheimer's. Nearly 35% of approved drugs implement their therapeutic effects by selectively interacting with specific GPCRs.
GPCRs are also called heptahelical, 7TM, or...
GPCRs are also called heptahelical, 7TM, or...