Dissection of the Role of VIMP in Endoplasmic Reticulum-Associated Degradation of CFTR\u0394F508

Xia Hou,Hong Jiang,Carthic Rajagopalan,Youming Xie,Fei Sun,Hongguang Wei,Qingtian Wu,Khalequz Zaman

doi:10.1038/s41598-018-23284-8

Abstract

Endoplasmic reticulum (ER)-associated protein degradation (ERAD) is an important quality control mechanism that eliminates misfolded proteins from the ER. The Derlin-1/VCP/VIMP protein complex plays an essential role in ERAD. Although the roles of Derlin-1 and VCP are relatively clear, the functional activity of VIMP in ERAD remains to be understood. Here we investigate the role of VIMP in the degradation of CFTRΔF508, a cystic fibrosis transmembrane conductance regulator (CFTR) mutant known to be a substrate of ERAD. Overexpression of VIMP markedly enhances the degradation of CFTRΔF508, whereas knockdown of VIMP increases its half-life. We demonstrate that VIMP is associated with CFTRΔF508 and the RNF5 E3 ubiquitin ligase (also known as RMA1). Thus, VIMP not only forms a complex with Derlin-1 and VCP, but may also participate in recruiting substrates and E3 ubiquitin ligases. We further show that blocking CFTRΔF508 degradation by knockdown of VIMP substantially augments the effect of VX809, a drug that allows a fraction of CFTRΔF508 to fold properly and mobilize from ER to cell surface for normal functioning. This study provides insight into the role of VIMP in ERAD and presents a potential target for the treatment of cystic fibrosis patients carrying the CFTRΔF508 mutation.

Highlights

Cystic fibrosis (CF) is one of the most common genetic diseases in the USA
To determine the involvement of VIMP in ERAD of CFTRΔF508, we examined if VIMP interacts with CFTRΔF508 by co-immunoprecipitation (IP)/immunoblotting analysis
We found that CFTRΔF508 was co-immunoprecipitated with VIMP by an anti-VIMP antibody but not by a pre-immune normal IgG (Fig. 1A, lanes 2, 3)

Summary

Introduction

Cystic fibrosis (CF) is one of the most common genetic diseases in the USA. CF is caused by mutations of the cystic fibrosis transmembrane conductance regulator (CFTR) gene[15]. Immunoblotting analysis showed that overexpression of VIMP reduced the steady-state level of immature CFTRΔF508 (“band B”) by 60% (Fig. 2A,B). A VIMP-targeting shRNA vector was transfected into CFBE41o− cells expressing CFTRΔF508. Immunoblotting analysis showed that the steady-state level of CFTRΔF508 was substantially increased by knockdown of VIMP (Fig. 2F, left panels).

Results

Conclusion