Trafficking-deficient G572R-hERG and E637K-hERG activate stress and clearance pathways in endoplasmic reticulum.
Trafficking-deficient G572R-hERG and E637K-hERG activate stress and clearance pathways in endoplasmic reticulum.
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DOI：
10.1371/journal.pone.0029885
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发表时间：
2012
期刊：
影响因子：
3.7
通讯作者：
Lian J
中科院分区：
文献类型：
--
作者：
Wang Y;Huang X;Zhou J;Yang X;Li D;Mao H;Sun HH;Liu N;Lian J
长QT综合征2型(LQT2)是所有长QT综合征中第二常见的类型。众所周知，运输缺陷突变的人类乙醚相关基因(HERG)蛋白经常与LQT2有关。细胞通过激活未折叠蛋白应答(UPR)和激活转录因子(ATF6)来应答错误折叠和运输缺陷的蛋白质，ATF6已被确定为哺乳动物UPR的关键调节因子。在本研究中，我们研究了内质网伴侣蛋白(钙粘蛋白和钙网蛋白)在G572R-HERG和E637K-HERG突变蛋白加工过程中的作用。将pcDNA3-WT-Herg、pcDNA3-G572R-Herg和pcDNA3-E637K-Herg分别导入U2OS和HEK293细胞。用激光共聚焦显微镜和免疫印迹技术分析细胞定位和蛋白表达。用免疫共沉淀法检测WT或突变体hERGs与Calnexin/Calreticrin的相互作用。为探讨泛素蛋白酶体途径在突变的HERG蛋白降解中的作用，用蛋白酶体抑制剂处理HEK293细胞，检测其对WT和突变的HERG蛋白稳态蛋白水平的影响。我们的结果表明，核心糖基化的未成熟形式G572R-HERG和E637K-HERG与钙调素和钙网蛋白相关的水平高于WT-HERG。两个突变的Herg蛋白都可以通过上调活性ATF6的水平来激活UPR。此外，蛋白酶体抑制增加了核心糖基化的未成熟形式的WT和突变型hERGs的水平。此外，与WT-HERG相比，蛋白酶体抑制后突变体hERGs与Calnexin/Calreticrin之间的相互作用更强。这些结果表明，运输缺陷的G572R-HERG和E637K-HERG突变蛋白可以激活内质网应激途径，并针对蛋白酶体进行降解。钙调素和钙网蛋白在这些过程中起着重要的作用。
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. 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. 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.
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影响因子：
37.8
作者：
Anderson, CL;Delisle, BP;January, CT
通讯作者：
January, CT
DOI：
10.1152/ajpheart.00052.2004
发表时间：
2004-08-01
影响因子：
4.8
作者：
Gong, QM;Anderson, CL;Zhou, ZF
通讯作者：
Zhou, ZF
影响因子：
6.1
作者：
Steiner, H;Winkler, E;Haass, C
通讯作者：
Haass, C
影响因子：
56.9
作者：
TRUDEAU, MC;WARMKE, JW;ROBERTSON, GA
通讯作者：
ROBERTSON, GA
哺乳动物转录因子 ATF6 以跨膜蛋白的形式合成，并在内质网应激时通过蛋白水解激活
影响因子：
3.3
作者：
Haze, K;Yoshida, H;Mori, K
通讯作者：
Mori, K