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Modulation of Ribosome Dynamics Rescues F508del CFTR Maturational Arrest

核糖体动力学调节挽救 F508del CFTR 成熟停滞

基本信息

批准号：
9051505
负责人：
Kathryn E Oliver
金额：
$ 2.54万
依托单位：
UNIVERSITY OF ALABAMA AT BIRMINGHAM
依托单位国家：
美国
项目类别：
财政年份：
2016
资助国家：
美国
起止时间：
2016-04-13 至 2016-12-31
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/9051505
关键词：
Address Amino Acids Apical Bicarbonates Biochemical Biogenesis Biological Assay Biological Models Birth Breeding Caucasians Cell Culture Techniques Cell model Cell surface Cells Chimera organism Chloride Ion Chlorides Critical Pathways Cystic Fibrosis Cystic Fibrosis Transmembrane Conductance Regulator Data Defect Development Diagnosis Disease Elongation Factor Endoplasmic Reticulum Epithelial Epithelium Foundations Future Gastrointestinal tract structure Harvest Histopathology Human Immunohistochemistry Individual Investigational Therapies Ion Transport Kinetics Knock-out Knockout Mice Lead Libraries Life Lung Transplantation Measurement Measures Mediating Messenger RNA Methods Modeling Molecular Chaperones Molecular Conformation Mus Mutation Nature North America Organ Pancreas Pathogenesis Pathway interactions Patients Peptide Synthesis Phenotype Phenylalanine Physiologic Monitoring Physiologic pulse Play Polyribosomes Population Positioning Attribute Printing Process Protein Conformation Proteins Publications Quality Control Reproductive system Respiratory System Respiratory tract structure Ribosomal Proteins Ribosomes Role Saccharomyces cerevisiae Safety Small Interfering RNA Structure Technology Testing Therapeutic Therapeutic Intervention Tissues Translations Transplant Recipients VX-770 VX-809 Western Blotting Work Yeasts airway epithelium clinically significant combinatorial cystic fibrosis patients flexibility foot gastrointestinal gastrointestinal system gene product genome-wide improved in vivo insight knock-down mouse model new therapeutic target novel novel strategies phenomics polypeptide protein degradation protein folding protein misfolding public health relevance research study respiratory response ribosome profiling small molecule stable cell line therapeutic target transcriptome sequencing

项目摘要

DESCRIPTION (provided by applicant): The most prevalent disease-associated mutation in cystic fibrosis (CF) is deletion of phenylalanine 508 (F508del) in the cystic fibrosis transmembrane conductance regulator (CFTR), which results in misprocessing and reduced apical localization of the protein in respiratory, gastrointestinal, pancreatic and other epithelia Our preliminary evidence demonstrates that suppression of ribosomal protein L12 (Rpl12) corrects the folding defect and functional cell surface expression of F508del CFTR. A number of recent and high-profile publications indicate that the ribosome not only regulates peptide synthesis and primary amino acid structure, but also plays a crucial "chaperone-like" role during protein folding [14-18]. Therefore, it is important to understand the mechanism by which Rpl12 mediates interaction with nascent F508del polypeptide during co- translational folding and test the feasibility of Rpl12 as a novel therapeutic target for individuals living with CF. Specific Ai 1: Characterize the effects of Rpl12 knockdown on F508del CFTR processing. Following siRNA-mediated Rpl12 inhibition, we will measure steady state levels of F508del expression, maturation, and function. In addition, we will determine whether abrogation of other ribosomal proteins located on the 60S subunit P stalk (in close proximity to Rpl12) also mediate rescue of F508del processing. Specific Aim 2: Ascertain the mechanism by which Rpl12 suppression rescues F508del CFTR function. We will use leading-edge technology (polysome analyses, ribosome profiling, RNA-Seq) to quantitatively address ribosome assembly, translation efficiency, elongation, and foot printing in response to Rpl12 knockdown. These studies will identify specific sub-domains within CFTR that are altered by deletion of F508, and test Rpl12 suppression as a means to overcome defects of this nature. Specific Aim 3: Determine in vivo relevance by development of RPL12 knockout, conditional knockout, or haploinsufficient mice. RPL12 mouse models will be cross-bred to CFTRF508del mice to assay for improvements in CF phenotyptic manifestations within the gastrointestinal and respiratory tracts. This will include studies of CFTR expression (biochemical analyses), histopathology, immunohistochemistry, and function (short circuit current measurements), and in vivo bioelectric measurements from the upper airways. Successful completion of the proposed experiments will: (1) establish a novel, specific ribosomal protein as a fundamental contributor to F508del CFTR folding and protein conformation, (2) demonstrate the in vivo role of ribosomal quality control during co-translational protein folding in the context of an important human disorder, and (3) provide new evidence for ribosomal-chaperone function during protein biogenesis. The in vivo experiments in mice will also furnish an important foundation for future studies aimed at modulating ribosome kinetics as an experimental therapeutic strategy for patients with this disease.