Small-molecule correctors divert CFTR-F508del from ERAD by stabilizing sequential folding states.
Small-molecule correctors divert CFTR-F508del from ERAD by stabilizing sequential folding states.
复制标题
小分子校正剂通过稳定顺序折叠状态将 CFTR-F508del 从 ERAD 中转移。
DOI：
10.1091/mbc.e23-08-0336
复制
发表时间：
2024
影响因子：
3.3
通讯作者：
Kopito,RonR
中科院分区：
文献类型：
--
作者：
Riepe,Celeste;Wąchalska,Magda;Deol,KirandeepK;Amaya,AnaisK;Porteus,MatthewH;Olzmann,JamesA;Kopito,RonR
超过80%的囊性纤维化（CF）患者携带囊性纤维化跨膜传导调节因子（CFTR）的F508del突变，CFTR是上皮细胞顶质膜（PM）上的氯离子通道。F508del损害CFTR折叠，导致其被内质网相关降解（ERAD）破坏。小分子纠正剂作为药物伴侣将CFTR-F508del从ERAD转移，是治疗CF的主要策略，但纠正剂的开发仍在继续，仅对ERAD如何靶向CFTR-F508del有初步的了解。我们进行了全基因组CRISPR/Cas9敲除筛选，以系统地鉴定CFTR-F508del ERAD的分子机制。尽管是er驻留的泛素连接酶，RNF5是E3中最受欢迎的，敲除outnf5只会适度降低CFTR-F508del的降解。在rnf5敲除背景下的亚库筛选发现RNF185是一个冗余连接酶，并证明cfr - f508del ERAD是稳健的。基因-药物相互作用实验表明，校正因子tezacaftor （VX-661）和elexacaftor （VX-445）稳定了序列的、抗rnf5的折叠状态。我们提出，将校正子结合到新生的CFTR-F508del上，通过稳定不是rnf5介导的泛素化底物的折叠状态来改变其折叠景观。
Over 80% of people with cystic fibrosis (CF) carry the F508del mutation in the cystic fibrosis transmembrane conductance regulator (CFTR), a chloride ion channel at the apical plasma membrane (PM) of epithelial cells. F508del impairs CFTR folding causing it to be destroyed by endoplasmic reticulum associated degradation (ERAD). Small-molecule correctors, which act as pharmacological chaperones to divert CFTR-F508del from ERAD, are the primary strategy for treating CF, yet corrector development continues with only a rudimentary understanding of how ERAD targets CFTR-F508del. We conducted genome-wide CRISPR/Cas9 knockout screens to systematically identify the molecular machinery that underlies CFTR-F508del ERAD. Although the ER-resident ubiquitin ligase, RNF5 was the top E3 hit, knocking outRNF5only modestly reduced CFTR-F508del degradation. Sublibrary screens in anRNF5knockout background identified RNF185 as a redundant ligase and demonstrated that CFTR-F508del ERAD is robust. Gene-drug interaction experiments illustrated that correctors tezacaftor (VX-661) and elexacaftor (VX-445) stabilize sequential, RNF5-resistant folding states. We propose that binding of correctors to nascent CFTR-F508del alters its folding landscape by stabilizing folding states that are not substrates for RNF5-mediated ubiquitylation.