人類第一型氯離子通道蛋白於細胞膜上蛋白質穩定性之調控

Protein stability of human CLC-1 channels at the plasma membrane

李宜靜(Yi-Ching Lee)

指導教授 ： 湯志永

國立臺灣大學/醫學院/生理學研究所/碩士(2017年)

https://doi.org/10.6342/NTU201702471

摘要

先天性肌肉強直症(myotonia congenita)是一種遺傳性的骨骼肌病變，是由於第七對染色體上的CLCN1基因發生突變，導致骨骼肌電壓敏感性氯離子通道CLC-1的功能改變。本實驗室先前利用yeast two-hybrid篩選技術，發現分子伴護蛋白FKBP8與泛素連接酶ZNRF1皆可以和CLC-1的C端區域產生交互作用。先前的實驗結果顯示FKBP8可能參與CLC-1在內質網的品質控管，進而提升CLC-1的總表現量和細胞膜上的表現量，至於FKBP8是否能夠直接穩定細胞膜上的CLC-1蛋白質表現則尚不清楚。另外，雖然ZNRF1被指出因為其N端具有豆蔻酸化(myristoylation)修飾，並且可以連結到細胞膜上，但ZNRF1是否會影響細胞膜上的CLC-1蛋白質表現也仍未確定。因此，本篇論文的目的，是想要探討FKBP8與ZNRF1是否可能參與CLC-1在細胞膜上的的品質控管。
首先，我們利用次細胞分群法(subcellular fractionation)去探討FKBP8在有無CLC-1的情況下的次細胞分布狀況。我們發現在與CLC-1共同表現的情形下，部分的FKBP8會從原本存在於內質網的分層轉為分布到細胞膜的分層。接著我們也利用差速離心法(differential centrifugation)觀察內質網、高基氏體及細胞膜蛋白質的分布趨勢。我們發現在與CLC-1共同表現的情形下，FKBP8在高基氏體及細胞膜的分布比例會明顯增加，也觀察到大量表現FKBP8時，CLC-1在內質網的分佈比例下降，而在細胞膜的分佈比例上升。由此推測，FKBP8可伴隨著CLC-1運出內質網，並一起經由高基氏體運送至細胞膜。
當我們利用shRNA壓制內生性ZNRF1的表現，會使CLC-1的蛋白質總表現量上升。當我們利用過氧化氫提升HEK293T細胞內生性ZNRF1活性時， CLC-1的總表現量也會明顯下降。此外，在有過氧化氫的情況下，大量表現ZNRF1則會進一步降低CLC-1的總表現量。我們還利用brefeldin A (BFA)來觀察細胞膜上CLC-1的代謝時程(turn over rate)，初步的結果顯示，利用Ub-K0干擾聚泛素鏈(polyubiquitin chain)的形成，會增加CLC-1於細胞膜上的穩定性。這些結果顯示，ZNRF1對細胞膜上CLC-1的降解效果會因細胞承受過氧化壓力而進一步提升，而且ZNRF1有可能經由對CLC-1催化聚泛素鏈的方式，促進細胞膜上的CLC-1降解。

關鍵字

人類第一型氯離子通道蛋白 ； 蛋白質穩定性 ； 細胞膜

並列摘要

Myotonia congenita is a hereditary skeletal muscle disease that is linked to mutations in the human CLCN1 gene, which encode the voltage-gated chloride channel CLC-1. Previously, through yeast two-hybrid screening, our lab demonstrated that cochaperone FKBP8 and E3 ligase ZNRF1 may interact with the C-terminal region of CLC-1. Previous data from our lab further showed that FKBP8 may be involved in the protein quality control system in endoplasmic reticulum (ER) and therefore increase both the total protein and the plasma membrane protein level of CLC-1. Whether FKBP8 could directly stabilize the protein level of CLC-1 at the plasma membrane remain unknown. On the other hand, although the E3 ubiquitin ligase ZNRF1may be anchored at the plasma membrane via N-myristoylation, it is still an open question whether ZNRF1 could regulate CLC-1 protein level at the plasma membrane. In this thesis, we aim to understand whether FKBP8 and ZNRF1 may involved in the protein quality control of CLC-1 at the plasma membrane.
First, we investigated the subcellular distribution of FKBP8 by using subcellular fractionation. We found that, when coexpressed with CLC-1, a significant fraction of FKBP8 was translocated from the endoplasmic reticulum fraction to the plasma membrane fraction. Next, by performing differential centrifugation, we examined the subcellular distribution of FKBP8 between endoplasmic reticulum and Golgi apparatus. We found that, upon coexpression with CLC-1, there was a significant increase in the distribution of FKBP8 in the Golgi apparatus fraction. Moreover, the same fractionation and centrifugation experiment also showed that coexpression with FKBP8 led to a notable enhamcement of the membrane localization of CLC-1. Thus, our data suggest that, together with CLC-1, FKBP8 may be exported out of endoplasmic reticulum and transported to Golgi apparatus and finally to plasma membrane.
shRNA suppression of endogenous ZNRF1expression resulted in significant upregelation of CLC-1 protein level. Activation of endogenous ZNRF1 activity with hydrogen peroxide led to notable reduction in CLC-1 protein level. Additionally, in the presence of hydrogen peroxide, coexpression with ZNRF1 significantly down-regulated CLC-1 protein expression. We also investigated the turn over rate of CLC-1 at the plasma membrane by using brefeldin A (BFA) treatment. We observed that overexpression of Ub-K0, which prevent the formation of polyubiquitin chain, seemed to stabilize CLC-1 protein level at the plasma membrane. Together, these results suggest that the degradation effect of ZNRF1 on CLC-1 is enhanced under oxidative stress, and that ZNRF1 may enhance protein degradation of CLC-1 at the plasma membrane by promoting CLC-1 polyubiquitination.

並列關鍵字

human CLC-1 channels ； protein stability ； plasma membrane

參考文獻

參考資料

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