Ribosomal perturbation as a mechanism to prevent misfolding of CFTR

核糖体扰动作为防止 CFTR 错误折叠的机制

基本信息

批准号：
10063541
负责人：
JOHN L HARTMAN
金额：
$ 55.62万
依托单位：
EMORY UNIVERSITY
依托单位国家：
美国
项目类别：
财政年份：
2017
资助国家：
美国
起止时间：
2017-12-15 至 2022-01-07
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/10063541
关键词：
Address Alleles Amino Acids Area Biochemical Biochemistry Biogenesis Biological Assay Birth Caucasians Cell surface Cells Clinical Code Codon Nucleotides Complex Cystic Fibrosis Cystic Fibrosis Transmembrane Conductance Regulator Data Defect Delta F508 mutation Development Disease Disease model Exhibits Genes Genetic Genetic Epistasis Genetic Polymorphism Goals Heterogeneity Human Immunohistochemistry Intervention Intestines Ion Transport Kinetics Knowledge Lead Length Libraries Life Mammalian Cell Measurement Mediating Mediator of activation protein Messenger RNA Modeling Molecular Molecular Chaperones Molecular Genetics Mus Mutation New Agents Other Genetics Pathogenesis Pathway interactions Patients Peptide Initiation Factors Peptides Pharmacology Phenotype Phenylalanine Physiology Positioning Attribute Protein Conformation Proteins Proteolysis Resolution Ribosomal Proteins Ribosomes Saccharomyces cerevisiae Safety Small Interfering RNA Speed Testing Tissues Trachea Transfer RNA Translating Translations Variant Work Yeasts airway epithelium base bioelectricity cell type cellular targeting clinically significant cold temperature conditional knockout cystic fibrosis patients disease phenotype disease-causing mutation efficacy evaluation experimental study gene product genome-wide human disease improved in vivo innovation knock-down loss of function mouse model multidisciplinary mutant novel novel strategies personalized medicine phenomics prevent protein folding protein misfolding ribosome profiling screening synergism trafficking transcriptome transcriptomics translational impact

项目摘要

Abstract Cystic Fibrosis (CF) is a common lethal autosomal recessive disorder, occurring in 1 per 2,500 to 3,500 U.S. births annually. The disease is caused by defects in the CF transmembrane conductance regulator (CFTR). Mutations that change the CFTR coding sequence alter integrity of peptide folding and lead to disease. In this project, we show that CFTR codon alterations—including both non-synonymous and synonymous polymorphisms on background of the common F508del variant—can perturb ribosome dynamics, consequent mRNA utilization, translational rate, and protein biogenesis. The critical relationship between protein folding and translational velocity is a topical area with ramifications ranging from basic CFTR trafficking to disease phenotype and intervention. Mechanistic underpinnings for this application are derived from genome-wide phenomic screening with the Saccharomyces cerevisiae deletion strain library to identify novel modulators of F508del CFTR maturation, leading to discovery of specific ribosomal protein modules that impact F508del CFTR folding. We have established that suppression of Rpl12, along with other 60S proteins comprising the ribosomal stalk, improve F508del CFTR processing and activity at the mammalian cell surface to levels (in primary airway epithelia) predicted to confer clinical benefit among CF patients and comparable in magnitude to lumacaftor, a new agent recently approved for this purpose. Our preliminary data indicate that improved folding and activity of F508del CFTR are attributable to effects on translational kinetics. We propose three Specific Aims to develop an innovative model relevant to CF pathogenesis: 1) Define ribosomal domains and functional pathway interactions that govern ∆F protein biogenesis, and expand the analysis to include other CFTR variants and complex alleles, 2) Ascertain the mechanism by which Rpl12 suppression rescues F508del CFTR function, including relevance of translation rate to disease causing CF mutations and other CFTR polymorphisms, 3) Determine in vivo significance by development of RPL12 conditional knockout or haplosufficient mice. Our team combines multidisciplinary and mutually reinforcing expertise in CFTR biochemistry, transport physiology, ribosome profiling, translational velocity, yeast phenomics, and molecular genetics to mechanistically address a fundamental hypothesis regarding ways the ribosome utilizes mRNA to influence folding and functional quality of resulting gene products. We will establish translation control as a novel and critical mediator of Yor1 and CFTR maturation, identify specific ribosome modules that influence the CFTR biogenesis pathway, and evaluate relevance and safety of repressing this target in a murine disease model. Such results will improve understanding of CF molecular mechanism, suggest novel approaches for personalized treatment of CF patients with the most common forms of the disease, and indicate clinical significance of an important new observation relevant to aberrant protein translation and a fatal genetic illness.

抽象的囊性纤维化 (CF) 是一种常见的致死性常染色体隐性遗传疾病，每 2,500 至 3,500 名美国人中就有 1 人患有囊性纤维化该疾病是由 CF 跨膜电导调节因子 (CFTR) 缺陷引起的。改变 CFTR 编码序列的突变会改变肽折叠的完整性并导致疾病。项目中，我们展示了 CFTR 密码子改变——包括非同义和同义常见 F508del 变体背景上的多态性 - 可能会扰乱核糖体动力学，从而导致 mRNA 利用、翻译率和蛋白质生物发生之间的关键关系。平移速度是一个热门领域，其影响范围从基本的 CFTR 贩运到疾病该应用的机制基础源自全基因组。使用酿酒酵母缺失菌株库进行表组筛选，以新鉴定调节剂 F508del CFTR 成熟，导致发现影响 F508del 的特定核糖体蛋白模块我们已经确定了 Rpl12 以及其他 60S 蛋白的抑制作用。核糖体柄，将哺乳动物细胞表面的 F508del CFTR 处理和活性提高到水平（在原发性气道上皮细胞）预计将为 CF 患者带来临床益处，且程度相当 lumacaftor是最近批准用于此目的的一种新药物，我们的初步数据表明，情况有所改善。 F508del CFTR 的折叠和活性可归因于对平移动力学的影响。我们提出了三种影响。具体目标是开发与 CF 发病机制相关的创新模型：1) 定义核糖体结构域和控制 ΔF 蛋白质生物发生的功能途径相互作用，并将分析扩展到包括其他 CFTR 变体和复杂等位基因，2) 确定 Rpl12 抑制拯救 F508del 的机制 CFTR 功能，包括翻译率与引起 CF 突变和其他 CFTR 疾病的相关性多态性，3) 通过开发 RPL12 条件敲除或确定体内意义我们的团队结合了 CFTR 领域的多学科和相辅相成的专业知识。生物化学、运输生理学、核糖体分析、翻译速度、酵母表型组学和分子遗传学来机械地解决关于核糖体利用 mRNA 的方式的基本假设影响所得基因产物的折叠和功能质量我们将建立翻译控制作为一种方法。 Yor1 和 CFTR 成熟的新颖且关键的介质，识别影响 Yor1 和 CFTR 成熟的特定核糖体模块 CFTR 生物发生途径，并评估在小鼠疾病中抑制该靶标的相关性和安全性这些结果将增进对 CF 分子机制的理解，并提出新的方法。对患有最常见疾病形式的 CF 患者进行个性化治疗，并指出临床与异常蛋白质翻译和致命遗传疾病相关的重要新观察的意义。