Functional Stability of Rescued ΔF508 Cystic Fibrosis Transmembrane Conductance Regulator in Airway Epithelial Cells
Functional Stability of Rescued ΔF508 Cystic Fibrosis Transmembrane Conductance Regulator in Airway Epithelial Cells
复制标题
DOI：
10.1165/rcmb.2008-0434oc
复制
发表时间：
2010-03-01
影响因子：
6.4
通讯作者：
Collawn, James F.
中科院分区：
文献类型：
--
作者：
Jurkuvenaite, Asta;Chen, Lan;Collawn, James F.
囊性纤维化（CF）跨膜传导调节因子（CFTR）基因中最常见的突变，Delta F508，导致产生快速降解的错误折叠蛋白。该突变蛋白质对温度敏感，之前的研究表明，低温拯救的通道对生理刺激的反应很差，并且在37摄氏度时从细胞表面迅速降解。在本研究中，我们测试了最近表征的药理学校正剂2-（5-氯-2-甲氧基苯基氨基）-4 '-甲基-[4，5' bithiazolyl-2 '-yl]-苯基-甲酮（corr-4a）对低温拯救的Delta F508 CFTR的细胞表面稳定性和功能的影响。我们证明，corr-4a显著增强了挽救的Delta F508 CFTR在37 ℃下长达12小时的蛋白质稳定性（P < 0.05）。利用萤火虫荧光素酶为基础的记者调查的机制，低温和corr-4a增强救援，我们发现，低温处理抑制蛋白酶体的功能，而corr-4a治疗抑制E1-E3泛素化途径。Ussing室研究表明，corr-4a在6小时时使cAMP介导的Delta F508 CFTR反应增加61%（P < 0.05），但在随后的时间点没有增加。然而，CFTR通道激活剂4-甲基-2-（5-苯基-1H-吡唑-3-基）-苯酚的加入显著增强了cAMP刺激的电流，表明生物化学可检测的细胞表面Delta F508 CFTR可以在正确的条件下被刺激。我们的研究表明，稳定挽救的Delta F508 CFTR不足以在气道上皮细胞中获得最大Delta F508 CFTR功能。这些结果强烈支持以下观点：Delta F508 CFTR的最大校正需要化学校正剂：（1）促进折叠并从内质网退出;（2）增强表面稳定性;和（3）改善通道活性。
The most common mutation in the cystic fibrosis (CF) transmembrane conductance regulator (CFTR) gene, Delta F508, results in the production of a misfolded protein that is rapidly degraded. The mutant protein is temperature sensitive, and prior studies indicate that the low-temperature-rescued channel is poorly responsive to physiological stimuli, and is rapidly degraded from the cell surface at 37 degrees C. In the present studies, we tested the effect of a recently characterized pharmacological corrector, 2-(5-chloro-2-methoxyphenylamino)-4'-methyl-[4,5'bithiazolyl-2'-yl]-phenyl-methanone (corr-4a), on cell surface stability and function of the low-temperature-rescued Delta F508 CFTR. We demonstrate that corr-4a significantly enhanced the protein stability of rescued Delta F508 CFTR for up to 12 hours at 37 degrees C (P < 0.05). Using firefly luciferase-based reporters to investigate the mechanisms by which low temperature and corr-4a enhance rescue, we found that low-temperature treatment inhibited proteasomal function, whereas corr-4a treatment inhibited the E1-E3 ubiquitination pathway. Ussing chamber studies indicated that corr-4a increased the cAMP-mediated Delta F508 CFTR response by 61% at 6 hours (P < 0.05), but not at later time points. However, addition of the CFTR channel activator, 4-methyl-2-(5-phenyl-1H-pyrazol-3-yl)-phenol, significantly augmented CAMP-stimulated currents, revealing that the biochemically detectable cell surface Delta F508 CFTR could be stimulated under the right conditions. Our studies demonstrate that stabilizing rescued Delta F508 CFTR was not sufficient to obtain maximal Delta F508 CFTR function in airway epithelial cells. These results strongly support the idea that maximal correction of Delta F508 CFTR requires a chemical corrector that: (1) promotes folding and exit from the endoplasmic reticulum; (2) enhances surface stability; and (3) improves channel activity.