D3 PharmaChemistry, Istituto Italiano di Tecnologia, Via Morego 30, 16163 Genova, Italy.
Cambridge Centre for Proteomics, Department of Biochemistry, University of Cambridge, Tennis Court Road, Cambridge CB2 1QR, UK.
Cells. 2022 Jun 16;11(12):1938. doi: 10.3390/cells11121938.
Cystic Fibrosis (CF) is a genetic disorder affecting around 1 in every 3000 newborns. In the most common mutation, F508del, the defective anion channel, CFTR, is prevented from reaching the plasma membrane (PM) by the quality check control of the cell. Little is known about how CFTR pharmacological rescue impacts the cell proteome.
We used high-resolution mass spectrometry, differential ultracentrifugation, machine learning and bioinformatics to investigate both changes in the expression and localization of the human bronchial epithelium CF model (F508del-CFTR CFBE41o-) proteome following treatment with VX-809 (Lumacaftor), a drug able to improve the trafficking of CFTR.
The data suggested no stark changes in protein expression, yet subtle localization changes of proteins of the mitochondria and peroxisomes were detected. We then used high-content confocal microscopy to further investigate the morphological and compositional changes of peroxisomes and mitochondria under these conditions, as well as in patient-derived primary cells. We profiled several thousand proteins and we determined the subcellular localization data for around 5000 of them using the LOPIT-DC spatial proteomics protocol.
We observed that treatment with VX-809 induces extensive structural and functional remodelling of mitochondria and peroxisomes that resemble the phenotype of healthy cells. Our data suggest additional rescue mechanisms of VX-809 beyond the correction of aberrant folding of F508del-CFTR and subsequent trafficking to the PM.
囊性纤维化（CF）是一种影响每 3000 名新生儿中就有 1 名的遗传疾病。在最常见的突变 F508del 中，有缺陷的阴离子通道 CFTR 被细胞的质量检查控制阻止到达质膜（PM）。目前对于 CFTR 药理学挽救如何影响细胞蛋白质组知之甚少。
我们使用高分辨率质谱、差速超离心、机器学习和生物信息学来研究 CF 模型（F508del-CFTR CFBE41o-）的人类支气管上皮细胞在用能够改善 CFTR 运输的药物 VX-809（Lumacaftor）治疗后蛋白质组的表达和定位的变化。
数据表明蛋白质表达没有明显变化，但检测到线粒体和过氧化物酶体蛋白的细微定位变化。然后，我们使用高内涵共聚焦显微镜进一步研究了这些条件下以及在患者来源的原代细胞中过氧化物酶体和线粒体的形态和组成变化。我们对数千种蛋白质进行了分析，并使用 LOPIT-DC 空间蛋白质组学方案确定了大约 5000 种蛋白质的亚细胞定位数据。
我们观察到 VX-809 的治疗诱导了线粒体和过氧化物酶体的广泛结构和功能重塑，类似于健康细胞的表型。我们的数据表明，除了纠正 F508del-CFTR 的异常折叠和随后向 PM 的运输之外，VX-809 还具有其他挽救机制。
