In vivo metabolic labeling of photoreceptor proteins

光感受器蛋白的体内代谢标记

• 批准号: 10612915
• 负责人: Sheila A Baker
• 金额: $ 19.31万
• 依托单位: UNIVERSITY OF IOWA
• 依托单位国家: 美国
• 财政年份: 2022
• 资助国家: 美国
• 起止时间: 2022-05-01 至 2025-04-30
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/10612915
• 关键词: Acyltransferase; Affinity Chromatography; Amino Acids; Amino Acyl-tRNA Synthetases; Azides; Biochemical; Biotin; Cell Differentiation process; Cells; Central Nervous System; Chemistry; Cone; Copper; Data; Data Collection; Development; Disease; Dissociation; Enterobacteria phage P1 Cre recombinase; Fluorescent Dyes; Future; Goals; Histology; Hour; Image; Knock-in Mouse; Label; Lead; LoxP-flanked allele; Mass Spectrum Analysis; Measures; Mediating; Metabolic; Methionine; Methionine-tRNA Ligase; Methods; Monitor; Mouse Strains; Mus; Neurons; Organelles; Photoreceptors; Phototransduction; Physiologic pulse; Population; Positioning Attribute; Protein Biosynthesis; Protein Dynamics; Protein Isoforms; Proteins; Proteome; Proteomics; Retina; Rod; Sensitivity and Specificity; Signal Transduction; Specificity; Structure; Synapses; Technology; Testing; Time; Tissues; Toxic effect; Transfer RNA Aminoacylation; Visualization; Work; cell type; comparative; experimental study; functional group; in vivo; interest; mutant; neural; novel; protein profiling; response; rho; sample collection; tool; unnatural amino acids

Project Summary The retina is often described as a relatively simple part of the central nervous system. Yet, it is composed of approximately 100 distinct cell types and we know very little about what differentiates these cell types from one another at the protein level. The problem with obtaining proteomic data restricted to a particular cell type is that the cell type of interest must be physically dissociated from its surrounding tissue before proteins can be extracted and identified. Attempts to do this invariably lead to cross contamination. A possible solution is offered by in vivo metabolic labeling, or BONCAT. In this approach non-canonical amino acids with azide functional groups are incorporated into protein. The azide then serves as a tag that can be labeled for visualization or affinity purification using click chemistry. In mice, it is possible to restrict the incorporation of the methionine surrogate, azidonorleucine (ANL), to select cell types. This is done through the Cre-dependent expression of a mutant methionyl- acyltransferase (MetRS*) that charges tRNA with ANL instead of methionine. Since only protein synthesized when ANL is present will be labeled, this provides both temporal and spatial control. The goal in this project is to adapt this technology to studies of photoreceptors as a test case for future retinal studies. Mice expressing Cre recombinase in either rods or cones will be crossed with MetRS* mice. We will validate these novel mouse lines, optimize ANL delivery, and test the specificity and sensitivity of click chemistry-mediated proteomic data collection. We expect that adapting this technology to the retina will open the door for both cell-type specific protein profiling and studies of dynamic protein changes in response to disease or various environmental stimulation.

项目摘要 视网膜通常被描述为中枢神经系统的相对简单部分。但是，是 由大约100种不同的细胞类型组成，我们对什么了解 在蛋白质水平上将这些细胞类型彼此区分。获得的问题 仅限于特定细胞类型的蛋白质组学数据是，感兴趣的细胞类型必须为 在提取蛋白质之前，物理与周围组织分离，并 确定。尝试这样做总是会导致交叉污染。一个可能的解决方案是 由体内代谢标签或Boncat提供。在这种方法中，非经典氨基酸 叠氮化物官能团被纳入蛋白质。叠氮化物然后用作标签 可以使用点击化学标记进行可视化或亲和力净化。在老鼠中，有可能 限制甲二氨酸替代偶氮蛋白酶（ANL）的掺入以选择细胞 类型。这是通过突变蛋白基的Cre依赖性表达来完成的 酰基转移酶（METRS*），用ANL代替蛋氨酸为TRNA充电。由于仅蛋白质 当出现ANL时合成的综合将标记，这同时提供时间和空间 控制。该项目的目标是使该技术适应感光器的研究 未来视网膜研究的案例。在杆或锥中表达CRE重组酶的小鼠将是 与metrs*小鼠交叉。我们将验证这些新型鼠标线，优化ANL输送， 并测试点击化学介导的蛋白质组学数据收集的特异性和灵敏度。我们 预计将这项技术适应视网膜将为两种细胞类型特定的门开门 蛋白质分析和动态蛋白质变化对疾病或各种的研究 环境刺激。