Department of Human Genetics, National Health Institute Doutor Ricardo Jorge, 1649-016 Lisboa, Portugal; University of Lisboa, Faculty of Sciences, BioISI-Biosystems & Integrative Sciences Institute, 1749-016 Lisboa, Portugal.
University of Lisboa, Faculty of Sciences, BioISI-Biosystems & Integrative Sciences Institute, 1749-016 Lisboa, Portugal.
J Biol Chem. 2019 Sep 6;294(36):13396-13410. doi: 10.1074/jbc.RA119.008738. Epub 2019 Jul 19.
Cystic fibrosis (CF) is a genetic disease caused by mutations in the gene encoding CF transmembrane conductance regulator (CFTR), a chloride channel normally expressed at the surface of epithelial cells. The most frequent mutation, resulting in Phe-508 deletion, causes CFTR misfolding and its premature degradation. Low temperature or pharmacological correctors can partly rescue the Phe508del-CFTR processing defect and enhance trafficking of this channel variant to the plasma membrane (PM). Nevertheless, the rescued channels have an increased endocytosis rate, being quickly removed from the PM by the peripheral protein quality-control pathway. We previously reported that rescued Phe508del-CFTR (rPhe508del) can be retained at the cell surface by stimulating signaling pathways that coax the adaptor molecule ezrin (EZR) to tether rPhe508del-Na/H-exchange regulatory factor-1 complexes to the actin cytoskeleton, thereby averting the rapid internalization of this channel variant. However, the molecular basis for why rPhe508del fails to recruit active EZR to the PM remains elusive. Here, using a proteomics approach, we characterized and compared the core components of wt-CFTR- or rPhe508del-containing macromolecular complexes at the surface of human bronchial epithelial cells. We identified calpain 1 (CAPN1) as an exclusive rPhe508del interactor that prevents active EZR recruitment, impairs rPhe508del anchoring to actin, and reduces its stability in the PM. We show that either CAPN1 down-regulation or its chemical inhibition dramatically improves the functional rescue of Phe508del-CFTR in airway cells. These observations suggest that CAPN1 constitutes an appealing target for pharmacological intervention, as part of CF combination therapies restoring Phe508del-CFTR function.
囊性纤维化（CF）是一种由编码 CF 跨膜电导调节因子（CFTR）的基因突变引起的遗传疾病，CFTR 是一种通常在上皮细胞表面表达的氯离子通道。最常见的突变导致 Phe-508 缺失，导致 CFTR 错误折叠和过早降解。低温或药理学校正剂可以部分挽救 Phe508del-CFTR 的加工缺陷，并增强这种通道变体向质膜（PM）的运输。然而，挽救的通道具有增加的内吞作用率，通过外周蛋白质量控制途径快速从 PM 中去除。我们之前报道过，通过刺激信号通路，可以将挽救的 Phe508del-CFTR（rPhe508del）保留在细胞表面，该信号通路诱使衔接分子 ezrin（EZR）将 rPhe508del-Na/H-交换调节因子-1 复合物系留在肌动蛋白细胞骨架上，从而避免这种通道变体的快速内化。然而，rPhe508del 无法招募活性 EZR 到 PM 的分子基础仍然难以捉摸。在这里，我们使用蛋白质组学方法，对人支气管上皮细胞表面含 wt-CFTR 或 rPhe508del 的大分子复合物的核心成分进行了表征和比较。我们鉴定出钙蛋白酶 1（CAPN1）为 rPhe508del 的独特相互作用蛋白，可防止活性 EZR 的募集，损害 rPhe508del 与肌动蛋白的锚定，并降低其在 PM 中的稳定性。我们表明，CAPN1 的下调或其化学抑制可显著改善气道细胞中 Phe508del-CFTR 的功能挽救。这些观察结果表明，CAPN1 可作为药物干预的有吸引力的靶点，作为恢复 Phe508del-CFTR 功能的 CF 联合治疗的一部分。
