title: Trafficking-deficient G572R-hERG and E637K-hERG activate stress and clearance pathways in endoplasmic reticulum.
abstract: BACKGROUND
  Long QT syndrome type 2 (LQT2) is the second most common type of all long QT syndromes. It is well-known that trafficking deficient mutant human ether-a-go-go-related gene (hERG) proteins are often involved in LQT2. Cells respond to misfolded and trafficking-deficient proteins by eliciting the unfolded protein response (UPR) and Activating Transcription Factor (ATF6) has been identified as a key regulator of the mammalian UPR. In this study, we investigated the role of ER chaperone proteins (Calnexin and Calreticulin) in the processing of G572R-hERG and E637K-hERG mutant proteins.
  
  
  METHODS
  pcDNA3-WT-hERG, pcDNA3-G572R-hERG and pcDNA3-E637K-hERG plasmids were transfected into U2OS and HEK293 cells. Confocal microscopy and western blotting were used to analyze subcellular localization and protein expression. Interaction between WT or mutant hERGs and Calnexin/Calreticulin was tested by coimmunoprecipitation. To assess the role of the ubiquitin proteasome pathway in the degradation of mutant hERG proteins, transfected HEK293 cells were treated with proteasome inhibitors and their effects on the steady state protein levels of WT and mutant hERGs were examined.
  
  
  CONCLUSIONS
  Our results showed that levels of core-glycosylated immature forms of G572R-hERG and E637K-hERG in association with Calnexin and Calreticulin were higher than that in WT-hERG. Both mutant hERG proteins could activate the UPR by upregulating levels of active ATF6. Furthermore, proteasome inhibition increased the levels of core-glycosylated immature forms of WT and mutant hERGs. In addition, interaction between mutant hERGs and Calnexin/Calreticulin was stronger after proteasome inhibition, compared to WT-hERG. These results suggest that trafficking-deficient G572R-hERG and E637K-hERG mutant proteins can activate ER stress pathways and are targeted to the proteasome for degradation. Calnexin and Calreticulin play important roles in these processes.
authors.affiliation: Ningbo Medical Center, LiHuiLi Hospital School of Medicine, Ningbo University, Ningbo, China.
authors.forename: Ying
authors.initials: Y
authors.lastname: Wang
authors.forename: Xiaoyan
authors.initials: X
authors.lastname: Huang
authors.forename: Jianqing
authors.initials: J
authors.lastname: Zhou
authors.forename: Xi
authors.initials: X
authors.lastname: Yang
authors.forename: Di
authors.initials: D
authors.lastname: Li
authors.forename: Haiyan
authors.initials: H
authors.lastname: Mao
authors.forename: Huan Huan
authors.initials: HH
authors.lastname: Sun
authors.forename: Ningsheng
authors.initials: N
authors.lastname: Liu
authors.forename: Jiangfang
authors.initials: J
authors.lastname: Lian
mesh_terms: D051702:Activating Transcription Factor 6 / Q000378:metabolism; D019943:Amino Acid Substitution / Q000235:genetics*; D037281:Calnexin / Q000378:metabolism; D037282:Calreticulin / Q000378:metabolism; D045744:Cell Line, Tumor; D000072237:ERG1 Potassium Channel; D004721:Endoplasmic Reticulum / Q000378:metabolism*; D059865:Endoplasmic Reticulum Stress; D051638:Ether-A-Go-Go Potassium Channels / Q000235:genetics* / Q000378:metabolism*; D006031:Glycosylation; D057809:HEK293 Cells; D006801:Humans; D018613:Microscopy, Confocal; D050505:Mutant Proteins / Q000378:metabolism*; D046988:Proteasome Endopeptidase Complex / Q000378:metabolism; D061988:Proteasome Inhibitors; D011485:Protein Binding; D021381:Protein Transport; D059748:Proteolysis; D015398:Signal Transduction; D025801:Ubiquitin / Q000378:metabolism
chemical_list: D051702:Activating Transcription Factor 6; D037282:Calreticulin; D000072237:ERG1 Potassium Channel; D051638:Ether-A-Go-Go Potassium Channels; C000606913:KCNH2 protein, human; D050505:Mutant Proteins; D061988:Proteasome Inhibitors; D025801:Ubiquitin; D037281:Calnexin; D046988:Proteasome Endopeptidase Complex