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：
10679562
负责人：
Julie Craft Van De Weghe
金额：
$ 24.9万
依托单位：
UNIVERSITY OF OKLAHOMA HLTH SCIENCES CTR
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：
2022-09-15 至 2025-08-31
项目状态：
未结题

来源：
https://reporter.nih.gov/project-details/10679562
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.

项目概要该项目的目标是确定纤毛病相关突变如何导致纤毛病理生理学。纤毛病是根源于纤毛功能障碍的疾病，并且表现出重叠的临床症状特征，包括发育迟缓、智力障碍、多指畸形、视网膜营养不良和进行性肾脏和肝脏受累。虽然纤毛病个体很少见，但总共有 1/500 的个体患有纤毛病，并且大约 30 种不同的纤毛病中的每一种都是由与纤毛相关的特定蛋白质网络功能障碍引起的，尽管精确的细胞机制仍然难以捉摸。初级纤毛是一个类似天线的突起几乎在每个细胞上都有发现；它从细胞体延伸出来，在那里接收和解释信号，从而允许细胞对环境做出反应。纤毛通过过渡区与细胞质分开调节蛋白质运输。专用的主动运输系统，鞭毛内运输，移动蛋白质跨越该屏障与纤毛结合，并与多种蛋白质保留方法结合使用选择性出口。参与这种选择性蛋白质运输的蛋白质涉及一系列纤毛病，表明异常的纤毛蛋白含量可能导致这些疾病的病因。我将使用我的新颖的人类纤毛分离方案和最先进的质谱方法来评估整体纤毛蛋白组成，确定对照组和患有纤毛病的细胞之间的差异- 相关的亚态突变。这项工作将提供一个全面、公正的错误定位目录 Joubert (K99) 和 Bardet-Biedl (R00) 综合征中的蛋白质，从而为未来的工作提供丰富的资源剖析与纤毛病有关的蛋白质网络。在补充方法中，我将确定如何纤毛病相关突变通过内源标记关键影响蛋白质运输的动态纤毛病蛋白并使用活细胞显微镜观察其运动。这项工作将回答关键问题关于纤毛病相关突变对纤毛进入、保留和退出的影响的问题。该应用提出了可轻松扩展到其他蛋白质/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态，而不是零动物模型中的突变。总之，这项工作将阐明纤毛病的病因并促进未来疗法的发展。