It has been reported that

It has been reported that heat shock (HS) assists both in the folding of newly synthesized proteins and the refolding of denatured proteins [10]. Molecular chaperones such as heat shock proteins (HSPs), including Hsp90 and Hsp70, induced by HS play an important role in the maturation of HERG [11,12]. In mammals, the heat shock factor (HSF) family consists of 4 subtypes and increases in response to HS to activate the transcription of molecular chaperones.
Materials and methods
Results
Discussion The novel HERG mutation A78T, found in a patient with LQT2, was located at the N-terminus of HERG proteins. The A78T-HERG protein failed to mature and was expressed as a highly ubiquitinated 135-kDa immature protein. Since ubiquitination of the protein to be degraded by the proteasome is known to occur in the core-glycosylated immature form at the ER [11,12], this finding indicates the impaired stability of A78T-HERG proteins to be degraded via the ubiquitin proteasome system. Protein maturation is facilitated by the accumulation of immature proteins (mass effect) as well as by posttranslational modifications including glycosylation. In the present study, MG132 increased the 17 alpha hydroxylase of the 135-kDa immature form of A78T-HERG, but not that of the 155-kDa mature form, suggesting that the accumulation of immature A78T-HERG did not facilitate its maturation. We previously demonstrated that MG132 increased the expression of the mature form of Kv1.5, a voltage-gated potassium channel, indicating that accumulated immature wild-type Kv1.5 protein could be converted to its mature form as a result of the mass effect [14]. Since a substantial portion of Kv1.5 is degraded via the ubiquitin–proteasome system, proteasomal inhibition results in the accumulation of a large amount of immature Kv1.5, facilitating its maturation as a mass effect. In contrast, during the synthesis of WT-HERG, most of the core-glycosylated immature protein is converted to the fully-glycosylated mature form [6]. Thus, accumulation of the immature form of HERG protein in a setting of proteasomal inhibition is lower than that of Kv1.5. The failure of MG132 to increase the expression of mature A78T-HERG suggests that accumulation of immature A78T-HERG is not sufficient to exert a mass effect, while accumulation of immature A78T-HERG may not increase its mature form because of severe dysfunction of its maturation process. Since the type and position of a mutation in the gene influence protein structure and stability [13], the A78T mutation may destabilize the HERG protein because of an alteration in the molecular radius or hydrophobicity of the amino acid at position 78. A78G-HERG yielded the mature protein, whereas A78V-HERG, similarly to A78T-HERG, did not. The former finding excludes the possibility that deletion of the methyl group by substitution for alanine destabilizes the A78T-HERG protein. The latter finding suggests that the larger molecular radius but not hydrophilicity of the threonine residue destabilizes A78T-HERG. Heat shock facilitates the folding of various proteins as mediated by the HSP family and can stabilize mutant proteins. HS has been reported to facilitate maturation of WT-HERG as well as mutant HERG by activating the HSF family [15]. HSF-1, 2, and 4 were reported to be expressed in humans. HSF-1 is a master regulator of Hsp70 and Hsp90. HSF-2 plays roles in the development of the brain and reproductive organs and acts as a major regulator of the proteostasis capacity against febrile-range thermal stress to prevent the accumulation of misfolded proteins [16]. HSF-4, which possesses transcriptional repressor properties in vivo, inhibits basal transcription of Hsp27 and Hsp90 in cultured cells [17]. In the present study, we found that HSF-1, but not HSF- 2 or 4, increased the expression of mature A78T-HERG protein. These results suggest that HSF-1 stabilizes A78T-HERG protein, promotes its transport to the plasma membrane, and increases the membrane current IKr. The ubiquitin proteasome system in cardiomyocytes, including HL-1 cells and neonatal rat cardiomyocytes, involves three activities: chymotrypsin-like, trypsin-like, and caspase-like activities. The degradation of several ion channel proteins showed similar time courses between cardiomyocytes and HEK293 cells, indicating no difference in ubiquitin–proteasome system activities in these cells. Thus, mutant HERG proteins such as A78T-HERG may be degraded through the ubiquitin–proteasome system in cardiomyocytes as well [18]. Accordingly, the clinical implications of our results are as follows. To date, many studies have shown that maturation of mutant HERG proteins can be improved by low temperature or HS and by application of E-4031 or a gastroprokinetic agent such as cisapride [19–21]. GGA is a non-toxic acyclic isoprenoid compound with a retinoid skeleton that increases Hsp70 expression by activating HSF-1 [22]. In the present study, GGA at the clinical concentration of 200nM facilitated maturation of the A78T-HERG protein, suggesting that GGA is of therapeutic value for patients with LQT2 caused by the A78T mutation in HERG.