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

• 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
• 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

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.

项目摘要（摘要） 囊性纤维化（CF）是由CF跨膜突变引起的致命常染色体隐性疾病 电导调节器（CFTR）。大多数CF患者至少有一份F508DEL-CFTR的副本 变体，导致蛋白质错误折叠和严重的多器官损伤。该提议的总体目标 是通过纠正导致的基本遗传缺陷来确定可以改善疾病表型的细胞靶标 从F508DEL和较少普遍的变体，例如过早的截断密码子（PTC）对当前的反应无反应 治疗。越来越明显的是，CFTR编码序列更改不仅破坏主要 蛋白质结构，但还扰动核糖体动力学，随之而来的mRNA利用和蛋白质 折叠/生物发生。在先前的研究中，酵母菌现象分析导致核糖体蛋白（RP）发现 模块作为F508DEL-CFTR贩运的效应子。在这种情况下，我们已经确定RPL12（UL11） 耗尽，通过降低翻译启动和伸长率来挽救F508DEL-CFTR缺陷 允许核糖体和/或相关的伴侣伴侣促进功能性蛋白质构象。发现 在当前的K99/R00中概述了RPL12抑制也纠正了稀有的PTC W1282X-CFTR， 在某种程度上可能使诊所中的患者受益。因此，我们假设RP沉默改变了翻译 速度和/或核糖体保真度，以部分挽救难治性CFTR变体的部分挽救合成和组装。我们 提出三个特定目的：（1）表征RP抑制对突变体CFTR生物发生的影响，（2） 确定RP沉默改变转化动力学以挽救难治性CFTR变体的机制， （3）确定转基因CF小鼠中RPL12破坏的体内相关性。我们将利用多学科 针对细胞生物学，生物化学，分子遗传学和哺乳动物生理学的专业知识 机械学上解决了关于核糖体影响蛋白质折叠的新方式的基本假设。 研究旨在将翻译控制作为CFTR期间的新颖和关键检查站建立。 处理，除了介导该途径的RPL12之外，还可以确定特定的RP。这样的结果将改善 了解囊性纤维化疾病机制，在动物模型中建立抑制RPL12的安全性，并 为在其他遗传性人类疾病状态中测试策略的相关性提供了基础。在资金期间 该奖项期间，奥利弗博士将接受CFTR生化技术，核糖体分析，RNA-的培训。 SEQ，生物信息学，CF的鼠模型以及职业/专业发展。这些领域的指导将 为她准备奖项的独立阶段（R00）阶段。埃默里大学提供了丰富的环境 在高度协作的学术研究中心，职业发展和利用最先进的设施。 一旦奥利弗博士成功完成了她的K99的研究，她就会有好处 担任教师职位和她作为独立CF研究人员的期望职业。