Elucidating premature translation termination in Cystic Fibrosis

阐明囊性纤维化中的过早翻译终止

• 批准号: 10362522
• 负责人: Christine Elizabeth Carbone
• 金额: $ 3.1万
• 依托单位: UNIV OF MASSACHUSETTS MED SCH WORCESTER
• 依托单位国家: 美国
• 财政年份: 2020
• 资助国家: 美国
• 起止时间: 2020-05-01 至 2023-04-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/10362522
• 关键词: Aminoglycosides; Binding; Biochemical; Biochemical Reaction; Biological Assay; Cells; Clinical Trials; Codon Nucleotides; Complex; Cryoelectron Microscopy; Cystic Fibrosis; Cystic Fibrosis Transmembrane Conductance Regulator; Data; Data Set; Defect; Disease; Fibrinogen; Gastrointestinal tract structure; Gene Mutation; Genes; Goals; Hydrolysis; In Vitro; Individual; Kinetics; Length; Liver; Lung; Measures; Mediating; Messenger RNA; Methods; Modeling; Molecular; Molecular Conformation; Nonsense Mutation; Nucleotides; Open Reading Frames; Pancreas; Peptides; Pharmaceutical Preparations; Poly(A)-Binding Proteins; Production; Proteins; RNA; RNA Biochemistry; Reaction; Regulator Genes; Resolution; Ribosomal Proteins; Ribosomes; Signal Transduction; Structure; System; Terminator Codon; Therapeutic; Time; Translations; Variant; base; cystic fibrosis patients; design; drug candidate; improved; inhibitor/antagonist; insight; method development; movie; mutant; nucleotide analog; peptidyl-tRNA; premature; prevent; recruit; release factor; termination factor; therapy design; treatment strategy

PROJECT SUMMARY A leading cause of Cystic Fibrosis (CF) is premature termination codons (PTCs) in the cystic fibrosis transmembrane conductance regulator (CFTR) gene. Suppression of translation termination at PTCs—i.e. PTC readthrough—to restore full-length CFTR protein may be a treatment strategy. Yet, current PTC readthrough drug candidates for CF are toxic (e.g. aminoglycosides) or ineffective (e.g. ataluren). Efficacy of PTC readthrough depends on efficiency of translation termination at the PTC. Thus, manipulating the molecular mechanisms of CFTR PTC termination to lower efficiency may improve PTC readthrough efficacy. However, strategies for such manipulations are limited in the absence of a detailed understanding of translation termination on CFTR PTCs. In the current model for normal termination, eukaryotic Release Factors 1 and 3 form a complex (eRF1•eRF3) that releases a newly synthesized protein from the ribosome. eRF1 recognizes a tetra-nucleotide stop codon at the end of an open reading frame, and catalyzes peptidyl-tRNA hydrolysis. Poly-A binding protein (PABP), which binds at 3′ ends of mRNA, recruits eRF3 and enhances termination efficiency. However, it remains unclear how PABP, eRF1, eRF3, and the tetra-nucleotide stop codon recognize the PTC to produce truncated CFTR protein. The goal of this proposal is to determine the biochemical and structural mechanism of translation termination. With guidance from Dr. Andrei Korostelev (expert in biochemical and structural mechanisms of translation), Dr. Allan Jacobson (expert in premature translation termination and PTC read-through), Dr. Phillip Zamore (RNA biochemist), Dr. Chen Xu (cryo-EM instrumentalist), and Dr. Nikolaus Grigorieff (expert in cryo-EM method development), release assays will be optimized to study the efficiency of translation termination mediated by eukaryotic release factors, and ensemble time-resolved (ENTIRE) cryo-EM will be used to capture structural intermediates of enzymatic reactions. Aim 1 will use defined mammalian translation systems to measure the individual effects of stop codon context, eRF1•eRF3, and PABP on the termination efficiencies (kcat/KM) of CFTR PTCs and the true stop codon. Aim 2 will visualize how the ribosome terminates on CFTR PTC G542X in its natural sequence context using ENTIRE cryo-EM. Collecting data at multiple time points will identify conformational changes and interactions between mRNA sequence, eRF1•eRF3, and PABP during termination. To reveal the termination mechanism on CFTR PTCs, structures and their progression intermediates will be compared with those recorded on the true CFTR stop codon. If successful, this study will reveal key molecular determinants of CFTR PTC termination, and may inform strategies to induce PTC readthrough for CF treatment.

项目概要 囊性纤维化 (CF) 的主要原因是囊性纤维化中的过早终止密码子 (PTC) 跨膜电导调节（CFTR）基因。抑制 PTC 处的翻译终止——即正温度系数 通读——恢复全长 CFTR 蛋白可能是一种治疗策略。然而，当前的 PTC 通读 CF 的候选药物是有毒的（例如氨基糖苷类）或无效的（例如 ataluren）。 PTC 通读的功效 取决于 PTC 翻译终止的效率。因此，操纵分子机制 CFTR PTC 终止以降低效率可能会提高 PTC 通读效率。然而，此类策略 由于缺乏对 CFTR PTC 翻译终止的详细了解，操作受到限制。 在当前的正常终止模型中，真核释放因子 1 和 3 形成复合体 (eRF1•eRF3) 从核糖体中释放出新合成的蛋白质。 eRF1 识别四核苷酸终止密码子 开放阅读框的末端，并催化肽基-tRNA 水解。聚 A 结合蛋白 (PABP)， 结合在 mRNA 3' 端，招募 eRF3 并提高终止效率。然而，目前尚不清楚如何 PABP、eRF1、eRF3 和四核苷酸终止密码子识别 PTC，产生截短的 CFTR 蛋白。 该提案的目标是确定翻译终止的生化和结构机制。 在 Andrei Korostelev 博士（翻译生化和结构机制专家）的指导下，博士。 Allan Jacobson（过早翻译终止和 PTC 通读专家）、Phillip Zamore 博士（RNA 生物化学家）、徐晨博士（冷冻电镜仪器专家）和 Nikolaus Grigorieff 博士（冷冻电镜方法专家） 开发），将优化释放测定以研究由 真核释放因子和整体时间分辨（ENTIRE）冷冻电镜将用于捕获结构 酶促反应的中间体。目标 1 将使用定义的哺乳动物翻译系统来测量 终止密码子上下文、eRF1•eRF3 和 PABP 对 CFTR 终止效率 (kcat/KM) 的个体影响 PTC 和真正的终止密码子。目标 2 将可视化核糖体如何在 CFTR PTC G542X 上终止 使用整个冷冻电镜的自然序列背景。在多个时间点收集数据将识别 终止过程中mRNA序列、eRF1•eRF3和PABP之间的构象变化和相互作用。 为了揭示 CFTR PTC 的终止机制，结构及其进展中间体将被 与真实 CFTR 终止密码子上记录的结果进行比较。如果成功，这项研究将揭示关键分子 CFTR PTC 终止的决定因素，并可能为 CF 治疗诱导 PTC 通读策略提供参考。