14-3-3蛋白質調控hErg鉀離子通道的生合成

Abstract

人類鉀離子通道hErg (human ether-à-go-go related gene) 的突變，會造成hErg鉀離子通道功能的低下，導致先天性第二型long QT症 (long QT syndrome type 2, LQT2) 的症狀。突變的hErg蛋白質通常有不正常的折疊 (misfolding)，和有增加內質網相關的蛋白質降解 (endoplasmic reticulum-associated degradation, ERAD) 作用，而使得鉀離子通道不易被運送至細胞膜表現。過去對於14-3-3蛋白質調控hErg鉀離子通道的研究結果主要著重於探討14-3-3蛋白質如何調控hErg鉀離子通道在細胞膜上的電生理功能，而對於14-3-3蛋白質是否會調控hErg蛋白質的生合成機制則並不清楚。為了瞭解這個問題，我們將hErg蛋白質表現在HEK293T細胞株，利用此細胞表現系統來探討14-3-3蛋白質對hErg蛋白質的生合成的調控機制。此外我們也利用表現hErg蛋白質的SH-SY5Y細胞株驗證在HEK293T細胞株得到的結果。我們發現HEK293T及SH-SY5Y細胞的hErg蛋白質的表現量在有14-3-3抑制劑BV02存在下會明顯減少。在SH-SY5Y細胞大量表現胜肽類的14-3-3抑制劑 (difopein) 也得到一致的結果。相反地，當在HEK293T細胞大量表現14-3-3蛋白質異形體 (isoforms): 14-3-3ε或14-3-3η，則會明顯增加hErg蛋白質表現量。半定量聚合酶連鎖反應實驗顯示hErg mRNA的量並沒有因14-3-3蛋白質的大量表現而受影響。後續在HEK293T細胞藉由酵素水解法去醣處理、brefeldin A (BFA) 抑制蛋白質運輸以及cycloheximide (CHX) 追蹤蛋白質降解等實驗方式，我們發現大量表現14-3-3蛋白質，主要是促進了hErg蛋白質在內質網的穩定度，而非影響了hErg蛋白質的運輸。我們也發現大量表現14-3-3蛋白質可能藉抑制E3 ubiquitin ligase功能而增加hErg蛋白質表現量。另一方面，在HEK293T細胞大量表現difopein，則是會造成hErg蛋白質膜運輸的缺陷，但幾乎不影響hErg蛋白質在內質網的穩定度。除此之外，在HEK293T細胞以forskolin活化PKA的訊息路徑，會有相似於大量表現14-3-3蛋白質的現象，即hErg蛋白質的表現量大幅增加。PKA活化導致的hErg表現量增加的效果，會被difopein抑制。綜而言之，我們的結果顯示14-3-3蛋白質具保護位於內質網的尚未成熟的hErg蛋白質的功能，而使hErg蛋白質免於被降解。且14-3-3蛋白質在活化的PKA訊息路徑下，會促進hErg蛋白質的成熟。

Congenital long QT syndrome type 2 (LQT2), caused by mutations of human ether-à-go-go related gene (hErg) K+ channel, is associated with hErg protein misfolding and enhanced endoplasmic reticulum (ER)-associated degradation, leading to defective membrane trafficking of the hErg K+ channel. The physiological function of cardiac hErg channel is subject to adrenergic regulation via either direct binding of cAMP or protein kinase A (PKA)-mediated phosphorylation, both of which are further potentiated by the interaction of hErg with 14-3-3 proteins. However, it remains unclear whether 14-3-3 proteins may also regulate the biogenesis of hErg protein. To address this question, we studied hErg protein expression in HEK293T cells and SH-SY5Y cells. Treatment with BV02, a non-peptidic 14-3-3 inhibitor can effectively decrease both the mature and immature forms of overexpressed hErg proteins in HEK293T cells and endogenous hErg proteins in SH-SY5Y cells. Besides, expression of difopein, a peptide inhibitor of 14-3-3s, also leads to the decrease of endogenous hErg protein expression in SH-SY5Y cells. In contrast, overexpression of 14-3-3ε or 14-3-3η isoforms enhances both the mature and immature forms hErg proteins in HEK293T cells. Semiquantitative RT-PCR analyses show that 14-3-3 overexpression does not affect the mRNA level of hErg. As revealed by glycosidase digestion, brefeldin A (BFA) treatment, and cycloheximide (CHX) chase experiments, overexpression of 14-3-3ε or 14-3-3η prominently increases protein stability of immature hErg at the ER, but has little influence on the ER-to-Golgi trafficking of the hErg K+ channel in HEK293T cells. Additionally, 14-3-3η overexpression may increase hErg expression by inhibiting E3 ubiquitin ligase mediated protein degradation. In comparison with expression of difopein causes defective membrane trafficking of hErg, while exerting negligible effect on hErg protein stability at the ER in HEK293T cells. Furthermore, similar to the effect of 14-3-3 overexpression, forskolin (FSK)-induced cAMP up-regulation, through the PKA signaling pathway, significantly promotes hErg expression level in HEK293T cells, an effect that can be abolished by difopein overexpression. Together, our results suggest that 14-3-3 proteins protect immature hErg from degradation at the ER, and facilitate PKA-mediated hErg protein maturation.