In vivo metabolic labeling of photoreceptor proteins

光感受器蛋白的体内代谢标记

基本信息

批准号：
10425524
负责人：
Sheila A Baker
金额：
$ 23.18万
依托单位：
UNIVERSITY OF IOWA
依托单位国家：
美国
项目类别：
财政年份：
2022
资助国家：
美国
起止时间：
2022-05-01 至 2024-04-30
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/10425524
关键词：
Acyltransferase Affinity Chromatography Amino Acids Amino Acyl-tRNA Synthetases Azides Biochemical Biotin Cell Differentiation process Cells Chemistry Cone Copper Data Data Collection Development Disease 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 Neuraxis Neurons Organelles Photoreceptors Phototransduction Physiologic pulse Population Positioning Attribute Problem Solving 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 Vertebrate Photoreceptors Visualization Work cell type comparative experimental study functional group in vivo interest mutant novel protein profiling relating to nervous system response rho sample collection tool unnatural amino acids

项目摘要

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.

项目概要视网膜通常被描述为中枢神经系统相对简单的部分。然而，它是由大约 100 种不同的细胞类型组成，我们对它们的组成知之甚少在蛋白质水平上区分这些细胞类型。获取的问题仅限于特定细胞类型的蛋白质组数据是感兴趣的细胞类型必须是在提取蛋白质之前与其周围组织物理分离确定。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记（BONCAT）提供。在这种方法中，非规范氨基酸具有叠氮化物官能团并入蛋白质中。然后叠氮化物作为标签可以使用点击化学进行标记以进行可视化或亲和纯化。在老鼠身上，有可能限制甲硫氨酸替代物叠氮正亮氨酸 (ANL) 的掺入，以选择细胞类型。这是通过突变甲硫氨酰的 Cre 依赖性表达来完成的酰基转移酶 (MetRS*)，用 ANL 而不是蛋氨酸装载 tRNA。因为只有蛋白质当 ANL 存在时合成的将被标记，这提供了时间和空间控制。该项目的目标是将这项技术应用于光感受器的研究作为测试未来视网膜研究的案例。在视杆细胞或视锥细胞中表达 Cre 重组酶的小鼠将与 MetRS* 小鼠杂交。我们将验证这些新型小鼠品系，优化 ANL 递送，并测试点击化学介导的蛋白质组数据收集的特异性和敏感性。我们预计将这项技术应用于视网膜将为细胞类型特异性打开大门蛋白质分析和研究响应疾病或各种情况的动态蛋白质变化环境刺激。