Role and mechanism of chaperones calreticulin and ERP57 in restoring trafficking to mutant HERG‑A561V protein

Abstract

Long QT syndrome type 2 is caused by a mutation in the human-ether-a-go-go-related gene (HERG) gene encoding the rapidly activating delayed rectifier K-current. HERG is a key cell membrane glycoprotein; however, whether the maturation process of HERG protein involves key molecules derived from the calnexin (cNX)/calreticulin (cRT) cycle and how these molecules work remains unknown. Using western blotting, the present study screened the key molecules CNX/CRT/endoplasmic reticulum protein 57 (ERP57) involved in this cycle, and it was revealed that the protein expression levels of cNX/cRT/ERP57 in wild-type (WT)/A561V cells were increased compared with those in WT cells (n=3; P<0.05). Additionally, a co-immunoprecipitation experiment was used to reveal that the ability of cNX/ERP57/cRT to interact with HERG was significantly increased in A561V and WT/A561V cells (n=3; P<0.05). A plasmid lacking the bb' domain of ERP57 was constructed and it was demonstrated that the key site of ERP57 binding to CRT and immature HERG protein is the bb' domain. The whole-cell patch-clamp technique detected that the tail current density increased by 46% following overexpression of cRT and by 53% following overexpression of ERP57 in WT/A561V cells. Overexpression of CRT and ERP57 could increased HERG protein levels on the membrane detected by confocal imaging. Furthermore, overexpression of ERP57 and cRT proteins could restore the HERG-A561V mutant protein trafficking process and rescue the dominant-negative suppression of WT. Overall, ERP57/CRT served a crucial role in the HERG-A561V mutant protein trafficking deficiency and degradation process.

The proteostasis interactomes of trafficking-deficient KV11.1 variants associated with Long QT Syndrome and pharmacological chaperone rescue

“…Through this approach, we identified several proteins already known to interact with K V 11.1, such as the E3 ubiquitin ligase ( NEDD4L) , the four-and-a-half LIM domains protein 2 ( FHL2) , the lectin-based folding chaperone calnexin ( CANX) , Caveolin-1 ( CAV1 ), and the 14-3-3ε protein ( YWHAE ). 32,33,39–42 The truncated variant interaction with 14-3-3ε was reduced compared to either WT or the full-length K V 11.1-G601S variant, consistent with previous work that suggests 14-3-3ε interacts with the C-terminal domain. 40 K V 11.1, a glyocoprotein, undergoes N-linked glycosylation at the N598 residue, and mutations at this site were shown to influence channel stability.…”

“…1C). 9,[32][33][34] Using manual annotation from Uniprot, we classified each protein interaction with a unique gene ontology (GO) term to represent the most prominent biological process (Supplementary Table 4).…”

The proteostasis interactomes of trafficking-deficient variants of the voltage-gated potassium channel KV11.1 associated with long QT syndrome

“…We identified several known K V 11.1 protein interactions such as 14-3-3ε ( YWHAE ) and calnexin ( CANX ) ( Fig. 1 C ) ( 9 , 26 , 27 , 28 ). Using manual annotation from UniProt, we classified each protein interaction with a unique gene ontology (GO) term to represent the most prominent biological process ( Table S4 ).…”

A quinolin-8-ol sub-millimolar inhibitor of UGGT, the ER glycoprotein folding quality control checkpoint

“…While other components of ERQC have been studied as drug targets, 6 , 7 , 8 cellular consequences of pharmacological modulation of UGGT have been relatively understudied—partly because of the risks associated with targeting core cell housekeeping machineries, and partly because there are no known UGGT selective inhibitors. UGGT is inhibited by its product uridine diphosphate ( UDP) 9 and squaryl derivatives of UDP 10 ; by the non-hydrolyzable UDP-Glucose (UDP-Glc) cofactor analog UDP-2-deoxy-2-fluoro-D-glucose (U2F); and by synthetic analogs of the N -linked Man 9 GlcNAc 2 glycan substrate, 11 , 12 but obviously none of these molecules are UGGT specific.…”