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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：
10463879
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/10463879
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.

项目概要（摘要）囊性纤维化（CF）是一种致死性常染色体隐性遗传病，由CF跨膜突变引起电导调节器（CFTR）。大多数CF患者携带至少一个F508 del-CFTR拷贝变异，导致蛋白质错误折叠和严重的多器官损伤。本提案的总体目标是目的是鉴定细胞靶点，通过纠正基本遗传缺陷，来自F508 del和不太普遍的变体，例如对当前的密码子不响应的过早截短密码子（PTC），疗法越来越明显的是，CFTR编码序列的改变不仅破坏了原发性蛋白质结构，但也扰乱核糖体动力学，随之而来的mRNA利用，和蛋白质折叠/生物成因。在以前的研究中，酵母表型分析导致核糖体蛋白（RP）的发现模块作为F508 del-CFTR贩运的效应器。在这种情况下，我们已经确定Rpl 12（uL 11）缺失通过降低翻译起始和延伸的速率来挽救F508 del-CFTR缺陷，允许核糖体和/或相关的分子伴侣促进功能性蛋白质构象。结果在本K99/R 00中概述的结果表明，Rp 112抑制还校正了罕见的PTC，W1282 X-CFTR，在一定程度上，这可能会使临床上的患者受益。因此，我们假设RP沉默改变了翻译，速度和/或核糖体保真度来部分拯救难治性CFTR变体的合成和组装。我们提出了三个具体目标：（1）表征RP抑制对突变CFTR生物发生的影响，（2）确定RP沉默改变翻译动力学以拯救难治性CFTR变体的机制，和（3）确定转基因CF小鼠中RPL 12破坏的体内相关性。我们将利用多学科细胞生物学、生物化学、分子遗传学和哺乳动物生理学方面的专业知识，机械地解决了一个关于核糖体影响蛋白质折叠的新方式的基本假设。这些研究的目的是建立翻译控制作为一种新的和关键的检查点，在CFTR 处理，并鉴定除了Rpl 12之外的介导该途径的特异性RP。这样的结果会有所改善了解囊性纤维化疾病机制，在动物模型中建立抑制Rpl 12的安全性，以及为测试该策略在其他遗传性人类疾病状态中的相关性提供基础。在融资期间在此期间，奥利弗博士将接受CFTR生物化学技术，核糖体分析，RNA- seq、生物信息学、CF小鼠模型和职业/专业发展。这些领域的指导将让她为独立（R 00）阶段的奖励做好准备。埃默里大学提供了丰富的环境，职业发展，并利用高度协作的学术研究中心的最先进的设施。一旦奥利弗博士成功完成了她的K99所描述的研究，她将处于有利地位，追求教师的位置和她理想的职业生涯作为一个独立的CF研究员。