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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：
10705217
负责人：
Julie Craft Van De Weghe
金额：
$ 22.95万
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/10705217
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 Dedications 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 Lipids Liver Mass Spectrum Analysis Mediating Membrane Membrane Lipids Mentors Methods Microtubules Movement Mutation Organelles Patients Persons Phase Physiology 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 cell fixing 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.

项目总结