Proteomics of Primary Cilia through Proximity Labeling

通过邻近标记研究初级纤毛的蛋白质组学

基本信息

批准号：
9590675
负责人：
Maxence V Nachury
金额：
$ 13.45万
依托单位：
UNIVERSITY OF CALIFORNIA, SAN FRANCISCO
依托单位国家：
美国
项目类别：
财政年份：
2015
资助国家：
美国
起止时间：
2015-12-01 至 2019-11-30
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/9590675
关键词：
Biochemical Biotin Biotinylation Catalogs Cells Cilia Collection Complex Cystic kidney DNA Sequence Alteration Data Defect Development Diffusion Disease Employee Strikes Environment Enzymes Erinaceidae Etiology Functional disorder G-Protein-Coupled Receptors Genes Genetic Transcription Genotype Goals Hereditary Disease Individual Integral Membrane Protein Interphase Cell Isotope Labeling Knowledge Label Lead Mammalian Cell Mass Spectrum Analysis Membrane Methods Mitochondria Molecular Analysis Molecular Models Mutation Names Obesity Pathway interactions Patients Phosphatidylinositols Phosphoric Monoester Hydrolases Phosphotransferases Pilot Projects Proteins Proteome Proteomics Receptor Signaling Research Retinal Degeneration SHH gene SSTR3 gene Sensory Signal Pathway Signal Transduction Signaling Molecule Smell Perception Somatostatin Sorting - Cell Movement Stimulus Technology Vision base ciliopathy comparative innovation insight malformation method development molecular modeling mutant novel novel therapeutic intervention public health relevance receptor response skeletal skeletal abnormality small molecule smoothened signaling pathway somatostatin receptor 3 tool trafficking

项目摘要

DESCRIPTION (provided by applicant): Primary cilia organize signaling pathways such as vision, olfaction and the Hedgehog developmental pathway inside a specialized environment. The soluble contents and the membrane of the cilium are separated from the rest of the cell by diffusion barriers, thus endowing cilia with a unique physico-chemical environment. Depending on the status of Hedgehog signaling, specific signaling intermediates move into or out of cilia and it is predicted that the ciliary composition is modified by Hedgehog signaling. However, it has not been possible to assess the extent of the changes in the ciliary proteome upon Hedgehog pathway activation because cilia cannot be readily isolated from mammalian cells. Dysfunction of primary cilia is the underlying cause of a collection of hereditary disorders named ciliopathies characterized by obesity, kidney cysts, skeletal malformations and sensory defects. Obtaining a catalogue of primary cilium proteins and comparing the ciliary proteome of ciliopathy to healthy cells would provide a rapid and effective way to characterize ciliopathies. Thus, proteomics of primary cilia is currently a major technical blockage in the discovery of novel ciliary mechanisms. Rather than isolating cilia, we sought to attach the small molecule biotin to all ciliary protein by targeting a biotinylation enzyme to the primary cilium. This approach named proximity labeling has shown great promise in obtaining the proteome of mitochondria. We present promising preliminary data showing the validity of this approach for ciliary proteomics and wish to apply it to compare the ciliary proteome between different signaling states or between ciliopathy mutants and healthy cells. The results obtained by comparative ciliary proteomics will provide unique insights into several important questions and generate testable molecular models. The proposal is intended to develop a promising technology and to serve as a stepping-stone towards a rapid and effective comparative ciliary proteomics platform.

描述（由适用提供）：初级纤毛组织信号通路，例如视觉，嗅觉和刺猬发育途径，内部的特殊环境中的刺猬发育途径。固体含量和纤毛的膜通过扩散屏障与其他细胞的其余部分分离，从而以独特的物理化学环境结束了纤毛。根据刺猬信号的状态，特定的信号传导中间体移入或退出纤毛，可以预测睫状成分是通过刺猬信号来修饰的。但是，由于无法轻易从哺乳动物细胞中分离出纤毛，因此无法评估刺猬途径激活后纤毛蛋白质组变化的程度。原发性纤毛的功能障碍是收集的遗传性疾病的根本原因，这些遗传性疾病称为ciliopathies，其特征是肥胖，肾脏囊肿，骨骼畸形和感觉缺陷。获得原代纤毛蛋白的目录并将纤毛病与健康细胞进行比较，将提供一种表征纤毛病的快速有效方法。这是原发性纤毛的蛋白质组学目前是发现新型睫状机制的主要技术阻塞。我们试图通过将生物素化酶靶向原代纤毛，而不是分离纤毛，而是试图将小分子生物素连接到所有睫状蛋白上。这种称为接近标记的方法在获得线粒体的蛋白质组方面表现出了巨大的希望。我们提供了有望的初步数据，显示了这种方法对睫状蛋白质组学的有效性，并希望将其应用于不同信号态或纤毛病变突变体和健康细胞之间的睫状蛋白质组学。通过比较睫状蛋白质组学获得的结果将为几个重要问题提供独特的见解，并产生可测试的分子模型。该提案旨在开发一种有前途的技术，并作为快速有效的比较睫状蛋白质组学平台的垫脚石。