Premchandar Aiswarya, Ming Ruiji, Baiad Abed, Da Fonte Dillon F, Xu Haijin, Faubert Denis, Veit Guido, Lukacs Gergely L
Department of Physiology, McGill University, Montréal, QC, Canada.
IRCM Mass Spectrometry and Proteomics Platform, Institut de Recherches Cliniques de Montréal, Montréal, QC, Canada.
Front Pharmacol. 2024 Apr 25;15:1389586. doi: 10.3389/fphar.2024.1389586. eCollection 2024.
Cystic fibrosis (CF) is a monogenic disease caused by mutations in the CF transmembrane conductance regulator () 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.
囊性纤维化（CF）是一种由囊性纤维化跨膜传导调节因子（CFTR）基因突变引起的单基因疾病。过早终止密码子（PTC）约占CF突变的9%，通常会导致CFTR阴离子通道出现严重的表达缺陷。尽管PTC作为遗传疾病的潜在病因很常见，但对其分子缺陷在转录本和蛋白质水平上的治疗敏感性的理解仍部分有待阐明。鉴于CF中的分子病理学取决于PTC的位置，需要多种药物干预来抑制加速的无义介导的mRNA衰变（NMD），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通读。G418诱导的通读结果以前仅在模拟局部序列背景对CFTR中PTC突变影响的报告模型中进行过研究。为了确定全长CFTR通读背景下PTC错误掺入的氨基酸及其比例，我们开发了一种亲和纯化（AP）-串联质谱（AP-MS/MS）流程。我们证实了在CFTR的G542X、R1162X和S1196X的UGA终止密码子处掺入了半胱氨酸、精氨酸和色氨酸残基。值得注意的是，我们观察到半胱氨酸和精氨酸的掺入比色氨酸更有利于这些CFTR PTC，这表明PTC附近以外的转录本序列和/或其他因素可以影响氨基酸掺入和全长CFTR功能表达。此外，确定通读CFTR PTC中错误掺入的氨基酸比例有助于通过优化CFTR调节剂组合来最大化PTC的功能挽救效率。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，并为完善针对各种导致CF的无义突变的突变依赖性治疗策略提供基础。
