Center for Biofunctional Molecules, Department of Chemistry, Yonsei University, Seoul 120-749, Korea.
J Am Chem Soc. 2011 Dec 21;133(50):20267-76. doi: 10.1021/ja206762p. Epub 2011 Nov 22.
Cystic fibrosis transmembrane conductance regulator (CFTR) is a cell-surface anion channel that permeates chloride and bicarbonate ions. The most frequent mutation of CFTR that causes cystic fibrosis is the deletion of phenylalanine at position 508 (ΔF508), which leads to defects in protein folding and cellular trafficking to the plasma membrane. The lack of the cell-surface CFTR results in a reduction in the lifespan due to chronic lung infection with progressive deterioration of lung function. Hsc70 plays a crucial role in degradation of mutant CFTR by the ubiquitin-proteasome system. To date, various Hsc70 inhibitors and transcription regulators have been tested to determine whether they correct the defective activity of mutant CFTR. However, they exhibited limited or questionable effects on restoring the chloride channel activity in cystic fibrosis cells. Herein, we show that a small molecule apoptozole (Az) has high cellular potency to promote membrane trafficking of mutant CFTR and its chloride channel activity in cystic fibrosis cells. Results from affinity chromatography and ATPase activity assay indicate that Az inhibits the ATPase activity of Hsc70 by binding to its ATPase domain. In addition, a ligand-directed protein labeling and molecular modeling studies also suggest the binding of Az to an ATPase domain, in particular, an ATP-binding pocket. It is proposed that Az suppresses ubiquitination of ΔF508-CFTR maybe by blocking interaction of the mutant with Hsc70 and CHIP, and, as a consequence, it enhances membrane trafficking of the mutant.
囊性纤维化跨膜电导调节因子（CFTR）是一种位于细胞表面的阴离子通道，可渗透氯离子和碳酸氢根离子。导致囊性纤维化的 CFTR 最常见突变是第 508 位苯丙氨酸缺失（ΔF508），这导致蛋白折叠缺陷和细胞向质膜的运输缺陷。由于慢性肺部感染导致肺功能进行性恶化，缺乏细胞表面 CFTR 会导致寿命缩短。热休克蛋白 70（Hsc70）在通过泛素-蛋白酶体系统降解突变型 CFTR 中起着至关重要的作用。迄今为止，已经测试了各种 Hsc70 抑制剂和转录调节剂，以确定它们是否能纠正突变型 CFTR 的缺陷活性。然而，它们在恢复囊性纤维化细胞中氯离子通道活性方面的效果有限或存在疑问。在此，我们表明，小分子凋亡诱导剂（Az）具有促进突变型 CFTR 膜转运及其在囊性纤维化细胞中氯离子通道活性的高细胞效力。亲和层析和 ATPase 活性测定的结果表明，Az 通过结合其 ATPase 结构域抑制 Hsc70 的 ATPase 活性。此外，配体定向蛋白标记和分子建模研究也表明 Az 结合到 ATPase 结构域，特别是结合到 ATP 结合口袋。据推测，Az 通过阻断突变与 Hsc70 和 CHIP 的相互作用来抑制 ΔF508-CFTR 的泛素化，从而增强突变体的膜转运。
