Grove Diane E, Rosser Meredith F N, Watkins Richard L, Cyr Douglas M
Department of Cell and Developmental Biology, School of Medicine, The UNC-Cystic Fibrosis Center, University of North Carolina, Chapel Hill, NC 27599, USA.
Methods Mol Biol. 2011;741:219-32. doi: 10.1007/978-1-61779-117-8_15.
Misfolding and premature degradation of F508del-CFTR is the major cause of cystic fibrosis. Components of the ubiquitin-proteasome system function on the surface of the endoplasmic reticulum to select misfolded proteins for degradation. The folding status of F508del-CFTR is monitored by at least two ER quality control checkpoints. The ER-associated Derlin-1/RMA1 E3 complex appears to recognize folding defects in CFTR that involve misassembly of NBD1 into a complex with the R-domain. In contrast, the cytosolic Hsp70/CHIP E3 complex appears to sense folding defects that occur after synthesis of NBD2. Herein we describe methods that allow for the study of how modulation of these ER quality control factors by siRNA impacts CFTR folding and degradation. The experimental system described employs transiently transfected HEK293 cells and is utilized to monitor the biogenesis of CFTR by both Western blot and pulse chase studies. Methods to detect complexes formed between CFTR folding intermediates and ER quality control factors will also be described.
F508del-CFTR的错误折叠和过早降解是囊性纤维化的主要原因。泛素-蛋白酶体系统的组分在内质网表面发挥作用，选择错误折叠的蛋白质进行降解。F508del-CFTR的折叠状态至少由两个内质网质量控制检查点监测。内质网相关的Derlin-1/RMA1 E3复合物似乎识别CFTR中涉及NBD1与R结构域错误组装形成复合物的折叠缺陷。相比之下，胞质Hsp70/CHIP E3复合物似乎感知NBD2合成后出现的折叠缺陷。在此我们描述了一些方法，这些方法可用于研究通过小干扰RNA对这些内质网质量控制因子的调控如何影响CFTR的折叠和降解。所描述的实验系统采用瞬时转染的HEK293细胞，并通过蛋白质免疫印迹和脉冲追踪研究来监测CFTR的生物合成。还将描述检测CFTR折叠中间体与内质网质量控制因子之间形成的复合物的方法。
