Activity-dependent probes for spatially-defined proteomics

用于空间定义的蛋白质组学的活性依赖性探针

• 批准号: 10686705
• 负责人: Christina Kim
• 金额: $ 141.43万
• 依托单位: UNIVERSITY OF CALIFORNIA AT DAVIS
• 依托单位国家: 美国
• 财政年份: 2023
• 资助国家: 美国
• 起止时间: 2023-09-01 至 2026-08-31
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/10686705
• 关键词: Alzheimer' s Disease; Animal Behavior; Behavioral; Benchmarking; Biochemical; Biological; Biological Markers; Brain; Brain Diseases; Cells; Communities; Cultured Cells; Disease model; Enzymes; Epilepsy; Gene Expression; Genetic; Genome; Goals; Hallucinogens; Health; Human; Label; Location; Methodology; Molecular; Mus; Neurons; Neurophysiology - biologic function; Neurosciences; Pattern; Pharmaceutical Preparations; Population; Property; Protein Engineering; Proteins; Proteome; Proteomics; Research; Spatial Distribution; Subgroup; Technology; Testing; Therapeutic; Translating; autism spectrum disorder; drug development; drug discovery; experience; high throughput screening; improved; insight; link protein; liquid chromatography mass spectrometry; neural; neuropsychiatric disorder; new therapeutic target; programs; protein expression; response; transcriptome

Project Summary Uncovering the molecular properties of functionally-defined neural ensembles is essential for understanding how these networks give rise to circuit function and animal behavior. This proposal aims to develop and deploy new molecular technologies that will enable the sub-cellular tagging and enrichment of proteins in activated subgroups of neurons in the brain. Linking protein expression to neural function at a large and unbiased scale is currently not possible with existing technologies. Thus the research program proposed here will fill a critical unmet gap in the molecular toolbox for neuroscientists. While prior technologies have focused on gaining genetic access to the genome or transcriptome of activated neurons, the approach here will identify the actual proteins expressed in specific subcellular compartments of functionally-relevant neurons. This is a key distinction, as gene expression alone cannot reveal to the actual physical location and expression patterns of translated proteins – which are the ultimate molecules that carry out the specific biochemical functions of our cells. To enable this goal, new activity-dependent proximity labeling probes will be developed using protein engineering. These probes will be improved and optimized through high-throughput screens performed in cultured cells, and then adapted for use in mammalian neurons. Concurrently, the probes will be tested and characterized in the mouse brain to improve and benchmark their function. To demonstrate their utility, the activity-dependent probes will be used to tag the proteins that are present in neurons undergoing high neural activity in response to a behavioral drug experience in mice. 5-MeO-DMT is a hallucinogenic drug that in humans has been associated with therapeutic potential for treating neuropsychiatric diseases. Neurons activated by 5-MeO-DMT will be labeled by the activity-dependent probes, and their spatial distribution throughout the brain will be examined using the fluorescent read-out of the new molecular enzyme. In addition, the probes will also tag the proteins that are present in these activated neurons, allowing the enrichment and unbiased profiling of these molecules using liquid chromatography mass spectrometry (proteomics). The sub- cellular proteome of these neurons will provide essential biological insight into the mechanism of hallucinogenic drugs, in addition to providing potential downstream targets for new drug discovery and development. More broadly, the new probes will be distributed freely to the neuroscience community to enable the study of protein expression in functionally-relevant populations of neurons.

项目摘要 揭示功能定义的神经合奏的分子特性对于理解 这些网络如何产生电路功能和动物行为。该建议旨在开发和部署 新的分子技术将使蛋白质在激活中的亚细胞标记和富集 大脑中神经元的亚组。将蛋白质表达与神经元的功能联系起来 现有技术目前不可能。这里提出的研究计划将充满关键 神经科学家的分子工具箱中的差距。虽然先前的技术专注于获得 遗传进入基因组或活化神经元的转录组，此处的方法将确定实际 在功能相关的神经元的特定亚细胞室中表达的蛋白质。这是关键 区别，因为仅基因表达无法揭示实际的物理位置和表达模式 翻译的蛋白质 - 这是执行我们的特定生化功能的最终分子 细胞。为了实现这一目标，将使用蛋白质开发新的活动依赖性邻近标签探针 工程。这些问题将通过执行的高通量屏幕改善和优化 培养的细胞，然后适应用于哺乳动物神经元。同时，问题将被测试，并且 在小鼠大脑中表征以改善和基准测试其功能。为了展示他们的效用 活动依赖性问题将用于标记存在高神经元中神经元中存在的蛋白质 响应小鼠行为药物体验的活性。 5-Meo-DMT是一种致幻药， 人类与治疗神经精神疾病的治疗潜力有关。神经元 由5-meo-dmt激活的活动依赖性问题将标记，它们的空间分布 通过新分子酶的荧光读数将检查大脑。此外， 这些问题还将标记这些活化神经元中存在的蛋白质，从而富集和 使用液相色谱质谱法（蛋白质组学）对这些分子的无偏分析。子 - 这些神经元的细胞蛋白质组将提供有关机制的基本生物学见解 致幻药，除了为新药发现和 发展。更广泛地，新问题将自由分发给神经科学社区以启用 在功能相关的神经元种群中蛋白表达的研究。