Characterization of the novel mutant A78T-HERG from a long QT syndrome type 2 patient: Instability of the mutant protein and stabilization by heat shock factor 1.
Characterization of the novel mutant A78T-HERG from a long QT syndrome type 2 patient: Instability of the mutant protein and stabilization by heat shock factor 1.
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DOI：
10.1016/j.joa.2015.10.005
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发表时间：
2016-10
影响因子：
2
通讯作者：
Yamamoto K
中科院分区：
文献类型：
--
作者：
Kondo T;Hisatome I;Yoshimura S;Mahati E;Notsu T;Li P;Iitsuka K;Kato M;Ogura K;Miake J;Aiba T;Shimizu W;Kurata Y;Sakata S;Nakasone N;Ninomiya H;Nakai A;Higaki K;Kawata Y;Shirayoshi Y;Yoshida A;Yamamoto K
人类ether-a-go-go相关基因（HERG）编码快速激活延迟整流钾通道的α亚基。该基因的突变导致长QT综合征2型（LQT 2）。在大多数情况下，突变降低了通道蛋白的稳定性，这可以通过热休克（HS）恢复。我们在LQT 2患者中鉴定了新的突变型A78 T-HERG。本研究的目的是表征这种突变蛋白，并测试HS和热休克因子（HSFs）是否可以稳定突变蛋白。在HEK 293细胞中表达A78 T-HERG和野生型HERG（WT-HERG），并通过免疫印迹、免疫沉淀、免疫荧光和全细胞膜片钳进行分析。当在HEK 293细胞中表达时，WT-HERG分别在135和155 kDa处产生不成熟和成熟形式的蛋白质。A78 T-HERG仅产生未成熟的形式，其被大量泛素化。蛋白酶体抑制剂MG 132增加了未成熟A78 T-HERG的表达，并增加了WT-HERG的未成熟和成熟形式。WT-HERG而非A78 T-HERG在质膜上表达。在全细胞膜片钳实验中，去极化脉冲在转染WT-HERG的细胞中诱发E4031敏感的HERG通道电流，但在转染A78 T-HERG的细胞中不诱发E4031敏感的HERG通道电流。A78 V突变体，而不是A78 G突变体，保持在类似于A78 T的未成熟形式。HS、HSF-1的表达或暴露于香叶基香叶基丙酮促进A78 T-HERG蛋白的成熟。A78 T-HERG的特征在于蛋白质不稳定性和质膜上的表达减少。突变体的稳定性被HSF-1部分恢复，表明HSF-1是治疗由HERG中A78 T突变引起的LQT 2的靶点。
The human ether-a-go-go-related gene (HERG) encodes the α-subunit of rapidly activating delayed-rectifier potassium channels. Mutations in this gene cause long QT syndrome type 2 (LQT2). In most cases, mutations reduce the stability of the channel protein, which can be restored by heat shock (HS). We identified the novel mutant A78T-HERG in a patient with LQT2. The purpose of the current study was to characterize this mutant protein and test whether HS and heat shock factors (HSFs) could stabilize the mutant protein. A78T-HERG and wild-type HERG (WT-HERG) were expressed in HEK293 cells and analyzed by immunoblotting, immunoprecipitation, immunofluorescence, and whole-cell patch clamping. When expressed in HEK293 cells, WT-HERG gave rise to immature and mature forms of the protein at 135 and 155 kDa, respectively. A78T-HERG gave rise only to the immature form, which was heavily ubiquitinated. The proteasome inhibitor MG132 increased the expression of immature A78T-HERG and increased both the immature and mature forms of WT-HERG. WT-HERG, but not A78T-HERG, was expressed on the plasma membrane. In whole-cell patch clamping experiments, depolarizing pulses evoked E4031-sensitive HERG channel currents in cells transfected with WT-HERG, but not in cells transfected with A78T-HERG. The A78V mutant, but not A78G mutant, remained in the immature form similarly to A78T. Maturation of the A78T-HERG protein was facilitated by HS, expression of HSF-1, or exposure to geranyl geranyl acetone. A78T-HERG was characterized by protein instability and reduced expression on the plasma membrane. The stability of the mutant was partially restored by HSF-1, indicating that HSF-1 is a target for the treatment for LQT2 caused by the A78T mutation in HERG.