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.

项目概要（摘要） 囊性纤维化（CF）是一种致命的常染色体隐性遗传疾病，由 CF 跨膜突变引起 电导调节器（CFTR）。大多数 CF 患者至少携带一份 F508del-CFTR 副本 变异，导致蛋白质错误折叠和严重的多器官损伤。该提案的总体目标 是确定可以通过纠正导致的基本遗传缺陷来改善疾病表型的细胞靶点 来自 F508del 和不太常见的变体，例如对当前的过早截断密码子 (PTC) 无反应 治疗。越来越明显的是，CFTR 编码序列的改变不仅破坏了原代 蛋白质结构，但也会扰乱核糖体动力学、随后的 mRNA 利用和蛋白质 折叠/生物发生。在之前的研究中，酵母表型组分析发现了核糖体蛋白 (RP) 模块作为 F508del-CFTR 贩运的效应器。在此背景下，我们确定 Rpl12 (uL11) 耗尽通过降低翻译起始和延伸率来挽救 F508del-CFTR 缺陷，从而 允许核糖体和/或相关分子伴侣促进功能性蛋白质构象。发现 目前 K99/R00 中概述的结果表明，Rpl12 抑制还可以纠正罕见的 PTC W1282X-CFTR， 到可能使临床患者受益的程度。因此，我们假设 RP 沉默会改变翻译 速度和/或核糖体保真度，以部分挽救难治性 CFTR 变体的合成和组装。我们 提出三个具体目标：（1）表征 RP 抑制对突变体 CFTR 生物发生的影响，（2） 确定 RP 沉默改变翻译动力学以拯救难治性 CFTR 变异的机制， (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 从机制上解决了有关核糖体影响蛋白质折叠的新方式的基本假设。 这些研究旨在将翻译控制确立为 CFTR 期间的一个新颖且关键的检查点 处理，并识别除 Rpl12 之外介导该途径的特定 RP。这样的结果将会改善 了解囊性纤维化疾病机制，建立动物模型中抑制 Rpl12 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。融资期间 在此奖项期间，Oliver 博士将接受 CFTR 生化技术、核糖体分析、RNA- seq、生物信息学、CF 小鼠模型以及职业/专业发展。这些领域的指导将 让她为奖项的独立 (R00) 阶段做好准备。埃默里大学为学生提供了丰富的环境 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 Oliver 博士成功完成 K99 描述的研究，她将能够很好地 追求教员职位和她作为独立 CF 研究员的理想职业。