Integrating functional proteomics, genome engineering, and live-cell microscopy to the study of ciliopathies

将功能蛋白质组学、基因组工程和活细胞显微镜集成到纤毛病的研究中

基本信息

批准号：
10550028
负责人：
Julie Craft Van De Weghe
金额：
$ 4.99万
依托单位：
UNIVERSITY OF WASHINGTON
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 2022-07-31
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/10550028
关键词：
Active Biological Transport Affect Animal Model Applications Grants Bardet-Biedl Syndrome Binding Proteins Biological Biology Brain Catalogs Cell Cycle Cell Line Cell Volumes Cell model Cells Chronic Cilia Clinical Clustered Regularly Interspaced Short Palindromic Repeats Complement Complex Congenital Abnormality Cues Cytoplasm Data Defect Development Developmental Delay Disorders Disease Distal Embryo Environment Etiology Exhibits Eye Functional disorder Future G-Protein-Coupled Receptors Genetic Genome engineering Goals Health Homeostasis Human Individual Intellectual functioning disability Joubert syndrome Kidney Knowledge Laboratories Libraries Light Lipids Liver Mass Spectrum Analysis Mediating Membrane Membrane Lipids Mentors Methods Microtubules Movement Mutation Organelles Patients Persons Phase Physiology Plant Roots Polydactyly Process Protein Analysis Protein Dynamics Protein Sortings Protein translocation Proteins Proteome Proteomics Protocols documentation Research Personnel Resources Retinal Dystrophy Ribosomes Role Signal Pathway Signal Transduction Skeleton Syndrome System Techniques Variant Work base career cellular imaging ciliopathy cilium biogenesis disease-causing mutation experimental study human disease innovation live cell microscopy member novel null mutation protein complex protein transport skeletal dysplasia skills targeted treatment trafficking

项目摘要

Project Summary The goal of this project is to determine how ciliopathy-related mutations contribute to ciliary pathophysiology. Ciliopathies are disorders rooted in ciliary dysfunction and exhibit overlapping clinical features, including developmental delay, intellectual disability, polydactyly, retinal dystrophy, and progressive involvement of the kidney and liver. While individually rare, ciliopathies combined affect 1/500 individuals and each of the ~30 distinct ciliopathies is caused by dysfunction of a specific protein network related to the cilium, although the precise cellular mechanisms remain elusive. The primary cilium is an antenna-like projection found on nearly every cell; it extends from the cell body, where it receives and interprets signals, thus allowing cells to respond to their environment. Cilia are partitioned from the cellular cytoplasm by the transition zone that regulates protein trafficking. A dedicated active transport system, intraflagellar transport, moves proteins bound for the cilium across this barrier and works in conjunction with multiple methods for protein retention and selective egress. The proteins involved in this selective protein transport are implicated in a range of ciliopathies, indicating that aberrant ciliary protein content likely contributes to the etiology of these disorders. I will use my novel human cilia isolation protocol and state-of-the-art mass spectrometry approach to assess global ciliary protein composition, defining differences between controls and cells harboring ciliopathy- associated hypomorphic mutations. This work will provide a comprehensive, unbiased catalog of mislocalized proteins in Joubert (K99) and Bardet-Biedl (R00) syndromes, thus providing a rich resource for future work to dissect the protein networks involved in ciliopathies. In a complementary approach, I will determine how ciliopathy-associated mutations affect the dynamics of protein trafficking by endogenously tagging key ciliopathy proteins and following their movement using live-cell microscopy. This work will answer critical questions about the impact of ciliopathy-associated mutations on entry into, retention within, and exit from cilia. This application proposes innovative techniques that are easily extendable to other proteins/ciliopathies, and importantly, investigates protein content and dynamics in the human disease context rather than with null mutations in animal models. Together, this work will shed light on the etiology of ciliopathies and catalyze the development of future therapies.

项目摘要该项目的目的是确定与纤毛病有关的突变如何促进睫状病理生理学。纤毛病是根植于睫状功能障碍的疾病，并且表现出重叠的临床功能，包括发育延迟，智力残疾，多态度，视网膜营养不良和进步肾脏和肝脏的参与。虽然是个性罕见的，但纤毛疗法总共影响了1/500个人，并且〜30个不同的纤毛病中的每一个都是由与纤毛相关的特定蛋白网络功能障碍引起的尽管精确的细胞机制仍然难以捉摸。主要纤毛是一种天线样投影在几乎每个细胞上都发现；它从接收和解释信号的细胞体延伸，从而允许细胞对其环境做出反应。纤毛由过渡区从细胞细胞质中分割调节蛋白质运输。专用的主动运输系统，flagellar运输，可移动蛋白质在此屏障中绑定了纤毛，并与多种蛋白质保留和选择性出口。与此选择性蛋白传输有关的蛋白质与一系列有关纤毛病，表明异常的睫状蛋白含量可能有助于这些疾病的病因。我将使用我的新型人类纤毛隔离方案和最先进的质谱法评估全球睫状蛋白组成，定义具有纤毛病的对照和细胞之间的差异相关的型肌肉突变。这项工作将提供一个全面的，无偏置的目录。 Joubert（K99）和Bardet-Biedl（R00）综合征的蛋白质，从而为将来的工作提供了丰富的资源剖析与纤毛病有关的蛋白质网络。在一种互补的方法中，我将确定如何纤毛病相关的突变通过内源标记钥匙影响蛋白质运输的动力学纤毛病蛋白并使用活细胞显微镜进行运动。这项工作将回答关键有关纤毛病相关突变对进入，内部保留和退出纤毛的影响的问题。该应用程序提出了易于扩展到其他蛋白质/纤毛病的创新技术，并且重要的是，研究人类疾病背景下的蛋白质含量和动力学，而不是无效动物模型中的突变。这项工作一起将阐明纤毛病的病因，并催化未来疗法的发展。