囊性纤维化(CF)是由CF跨膜传导调节因子(CFTR)基因突变引起的单基因疾病。过早终止密码子(PTC)代表约9%的CF突变,通常会导致CFTR阴离子通道的严重表达缺陷。尽管PTCs是遗传疾病的根本原因,了解其分子缺陷的治疗敏感性,在转录本和蛋白质水平上仍部分阐明。鉴于分子病理学取决于CF中的PTC位置,需要多种药物干预来抑制加速的无意义介导的mRNA衰减(NMD),为了纠正由错误掺入的氨基酸引起的CFTR构象缺陷,并增强低效的终止密码子连读。以前仅在模拟局部序列环境对CFTR中的PTC突变的影响的报告模型中研究了G418诱导的连读结果。为了在全长CFTR读入的情况下确定不掺入的氨基酸及其在PTC中的比例,我们开发了亲和纯化(AP)-串联质谱(AP-MS/MS)管道。我们确认了Cys的合并,Arg,和Trp残基在G542X的UGA终止密码子,R1162X,和S1196X在CFTR。值得注意的是,我们观察到Cys和Arg在这些CFTRPTC中的掺入比Trp更有利,这表明转录物序列超出PTC和/或其他因素的接近程度可以影响氨基酸掺入和全长CFTR功能表达。此外,通过优化CFTR调节剂组合,在通读CFTRPTC中建立错误掺入的氨基酸比例有助于使PTC的功能挽救效率最大化。总的来说,我们的发现有助于理解各种CFTR无义突变背后的分子缺陷,并为完善各种引起CF的无义突变的突变依赖性治疗策略提供了基础.
Cystic fibrosis (CF) is a monogenic disease caused by mutations in the CF transmembrane conductance regulator (CFTR) gene. Premature termination codons (PTCs) represent ∼9% of CF mutations that typically cause severe expression defects of the CFTR anion channel. Despite the prevalence of PTCs as the underlying cause of genetic diseases, understanding the therapeutic susceptibilities of their molecular defects, both at the transcript and protein levels remains partially elucidated. Given that the molecular pathologies depend on the PTC positions in CF, multiple pharmacological interventions are required to suppress the accelerated nonsense-mediated mRNA decay (NMD), to correct the CFTR conformational defect caused by misincorporated amino acids, and to enhance the inefficient stop codon readthrough. The G418-induced readthrough outcome was previously investigated only in reporter models that mimic the impact of the local sequence context on PTC mutations in CFTR. To identify the misincorporated amino acids and their ratios for PTCs in the context of full-length CFTR readthrough, we developed an affinity purification (AP)-tandem mass spectrometry (AP-MS/MS) pipeline. We confirmed the incorporation of Cys, Arg, and Trp residues at the UGA stop codons of G542X, R1162X, and S1196X in CFTR. Notably, we observed that the Cys and Arg incorporation was favored over that of Trp into these CFTR PTCs, suggesting that the transcript sequence beyond the proximity of PTCs and/or other factors can impact the amino acid incorporation and full-length CFTR functional expression. Additionally, establishing the misincorporated amino acid ratios in the readthrough CFTR PTCs aided in maximizing the functional rescue efficiency of PTCs by optimizing CFTR modulator combinations. Collectively, our findings contribute to the understanding of molecular defects underlying various CFTR nonsense mutations and provide a foundation to refine mutation-dependent therapeutic strategies for various CF-causing nonsense mutations.