Hsp90 prevents interaction between CHIP and HERG proteins to facilitate maturation of wild-type and mutant HERG proteins
Hsp90 prevents interaction between CHIP and HERG proteins to facilitate maturation of wild-type and mutant HERG proteins
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DOI：
10.1093/cvr/cvt200
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发表时间：
2013-12-01
影响因子：
10.8
通讯作者：
Hisatome, Ichiro
中科院分区：
文献类型：
--
作者：
Iwai, Chisato;Li, Peili;Hisatome, Ichiro
以HEK293细胞和HL-1小鼠心肌细胞为研究对象，利用Hsp90抑制剂、葛兰素(GA)和自由基以及Hsp90的过表达，研究Hsp90在野生型(WT)和突变型乙醚-a-GO-GO相关基因(HERG)表达和成熟中的作用。GA和自由基酚抑制Herg-FLAG蛋白的成熟，增加其不成熟形式。Hsp90的共表达可抵消Hsp90抑制剂的作用，抑制HERG蛋白的泛素化。过表达的Hsp90还抑制内源性Hsp70相互作用蛋白C末端(CHIP)与HERG-FLAG蛋白的结合。HSP90诱导的功能性HERG蛋白表达增加和HERG通道电流增强证实了HSP90诱导的HERG蛋白表达增加。在GA处理的细胞中，CHIP过表达减少了成熟和未成熟形式的Herg-FLAG蛋白。在HL-1细胞中，HSP90促进内源性ERG蛋白的成熟，而CHIP则减少这两种形式的ERG蛋白。含有致病错义突变的突变HERG蛋白主要以不成熟的形式存在，与WT相比具有更高的芯片结合能力；Hsp90的过表达抑制了这种联系。过表达的Hsp90增加了HERG(1122fs/147)蛋白的成熟形式，减少了其泛素化形式，增强了其在内质网和质膜上的免疫反应性，并增加了突变介导的膜电流。CHIP过表达可减少HERG(1122fs/147)蛋白的未成熟形式，通过抑制Hsp90的结合增强HERG蛋白的表达，可能成为治疗HERG蛋白转运异常所致的长QT综合征2的新策略。
We examined the role of Hsp90 in expression and maturation of wild-type (WT) and mutant ether-a-go-go related gene (HERG) proteins by using Hsp90 inhibitors, geldanamycin (GA) and radicicol, and Hsp90 overexpression.The proteins were expressed in HEK293 cells or collected from HL-1 mouse cardiomyocytes, and analysed by western blotting, immunoprecipitation, immunofluorescence, and whole-cell patch-clamp techniques. GA and radicicol suppressed maturation of HERG-FLAG proteins and increased their immature forms. Co-expression of Hsp90 counteracted the effects of Hsp90 inhibitors and suppressed ubiquitination of HERG proteins. Overexpressed Hsp90 also inhibited the binding of endogenous C-terminus of Hsp70-interacting protein (CHIP) to HERG-FLAG proteins. Hsp90-induced increase of functional HERG proteins was verified by their increased expression on the cell surface and enhanced HERG channel currents. CHIP overexpression decreased both mature and immature forms of HERG-FLAG proteins in cells treated with GA. Hsp90 facilitated maturation of endogenous ERG proteins, whereas CHIP decreased both forms of ERG proteins in HL-1 cells. Mutant HERG proteins harbouring disease-causing missense mutations were mainly in the immature form and had a higher binding capacity to CHIP than the WT; Hsp90 overexpression suppressed this association. Overexpressed Hsp90 increased the mature form of HERG(1122fs/147) proteins, reduced its ubiquitinated form, increased its immunoreactivity in the endoplasmic reticulum and on the plasma membrane, and increased the mutant-mediated membrane current. CHIP overexpression decreased the immature form of HERG(1122fs/147) proteins.Enhancement of HERG protein expression through Hsp90 inhibition of CHIP binding might be a novel therapeutic strategy for long QT syndrome 2 caused by trafficking abnormalities of HERG proteins.