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Modulation of ribosome velocity as a means to rescue refractory CF-causing variants

调节核糖体速度作为拯救难治性 CF 引起的变异的一种手段

基本信息

批准号：
10445444
负责人：
Kathryn E Oliver
金额：
$ 24.9万
依托单位：
EMORY UNIVERSITY
依托单位国家：
美国
项目类别：
财政年份：
2021
资助国家：
美国
起止时间：
2021-09-01 至 2024-08-31
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/10445444
关键词：
Address Advisory Committees Alleles Amino Acid Sequence Animal Model Animals Anions Area Award Basic Science Biochemical Biochemistry Biogenesis Bioinformatics Career Mobility Cell Line Cell membrane Cells Cellular biology Clinic Code Codon Nucleotides Colon Coupled Cystic Fibrosis Cystic Fibrosis Transmembrane Conductance Regulator DNA Sequence Alteration Data Defect Delta F508 mutation Development Diagnosis Disease Disease model Environment Epithelial Exhibits Faculty Fellowship Funding Genetic Goals Heart Human Immunohistochemistry Inbred F344 Rats Individual Inherited Intervention Intestines Ion Transport Kinetics Knowledge Label Lead Libraries Measures Mediating Mentors Mentorship Messenger RNA Modeling Molecular Molecular Chaperones Molecular Genetics Monitor Mus Mutation Nasal Epithelium Nonsense-Mediated Decay Nucleotides Organoids Pancreas Participant Pathway interactions Patients Pharmaceutical Preparations Pharmacology Phase Phenotype Phenylalanine Physiology Positioning Attribute Postdoctoral Fellow Protein Biosynthesis Protein Conformation Protein Inhibition Proteins Refractory Research Research Personnel Resolution Ribosomal Proteins Ribosomes Safety Severities Small Interfering RNA Techniques Terminator Codon Testing Thyroid Gland Tissues Training Transgenic Organisms Translation Initiation Translational Research Translations United States National Institutes of Health Universities Variant Work Yeasts airway epithelium base bronchial epithelium career cellular targeting clinical heterogeneity clinical phenotype cystic fibrosis mouse cystic fibrosis patients disease phenotype disease-causing mutation experience experimental study genome-wide human disease ileum improved in vivo knock-down knowledge base mRNA Stability member mouse model multidisciplinary multiorgan damage mutant novel patient population phenomics premature programs protein degradation protein folding protein misfolding response ribosome profiling screening trafficking transcriptome sequencing

项目摘要

PROJECT SUMMARY (ABSTRACT) Cystic fibrosis (CF) is a lethal autosomal recessive disorder caused by mutation of the CF transmembrane conductance regulator (CFTR). The majority of CF patients harbor at least one copy of the F508del-CFTR variant, which results in protein misfolding and severe multi-organ damage. An overarching goal of this proposal is to identify cellular targets that can ameliorate disease phenotype by correcting basic genetic defects resulting from F508del and less prevalent variants, such as premature truncation codons (PTCs) unresponsive to current therapy. It has become increasingly evident that CFTR coding sequence alterations not only disrupt primary protein structure, but also perturb ribosome dynamics, consequent mRNA utilization, and protein folding/biogenesis. In previous studies, yeast phenomic analyses led to discovery of ribosomal protein (RP) modules as effectors of F508del-CFTR trafficking. In this context, we have established that Rpl12 (uL11) depletion rescues the F508del-CFTR defect by reducing rates of translation initiation and elongation, thereby allowing the ribosome and/or associated chaperones to promote a functional protein conformation. Findings outlined in the present K99/R00 demonstrate that Rpl12 suppression also corrects a rare PTC, W1282X-CFTR, to a degree that may benefit patients in the clinic. Thus, we hypothesize that RP silencing alters translational velocity and/or ribosome fidelity to partially rescue synthesis and assembly of refractory CFTR variants. We propose three specific aims: (1) characterize the effect(s) of RP inhibition on mutant CFTR biogenesis, (2) ascertain the mechanism by which RP silencing alters translational kinetics to rescue refractory CFTR variants, and (3) determine in vivo relevance of RPL12 disruption in transgenic CF mice. We will utilize multidisciplinary expertise directed towards cellular biology, biochemistry, molecular genetics, and mammalian physiology to mechanistically address a fundamental hypothesis regarding new ways the ribosome influences protein folding. The studies are intended to establish translation control as a novel and critical checkpoint during CFTR processing, and identify specific RPs in addition to Rpl12 that mediate this pathway. Such results will improve understanding of cystic fibrosis disease mechanism, establish safety of repressing Rpl12 in animal models, and provide a basis for testing relevance of the strategy in other inherited human disease states. During the funding period of this award, Dr. Oliver will receive training in CFTR biochemical techniques, ribosome profiling, RNA- seq, bioinformatics, murine models of CF, and career/professional development. Mentorship in these areas will prepare her for the independent (R00) phase of the award. Emory University provides a rich environment for career advancement and leverages state-of-the-art facilities in a highly collaborative academic research center. Once Dr. Oliver has successfully completed the studies described for her K99, she will be well positioned to pursue a faculty position and her desired career as an independent CF researcher.

项目总结（摘要）