分子伴護蛋白調控人類第一型氯離子通道蛋白之品質控管

Abstract

先天性肌肉強直症 (myotonia congenita) 是一種遺傳性肌肉疾病，是由於電壓敏感性氯離子通道CLC-1基因發生突變所造成。A531V是一種先天性肌肉強直症的突變型，其開關特性 (gating property) 與野生型 (wild-type) 並無明顯差別；但A531V蛋白質的表現量卻明顯較少，此表現減少的原因之一是由於蛋白酶體降解增加。本實驗室先前的研究發現A531V與伴護蛋白Hsp90的輔助伴護蛋白Aha1以及帶有PPIase功能的伴護蛋白 FKBP8有交互作用。分子伴護蛋白可協助蛋白質的折疊，以及將不具功能、錯誤摺疊的蛋白經由降解途徑移除。本篇論文目的為檢測可能調控CLC-1的chaperon，並且了解 CLC-1 WT和A531V是否經由Hsp70-Hsp90伴護蛋白系統來調控其蛋白質的品質控管。 我們發現大量表現Aha1可增加CLC-1 WT與A531V的穩定性，而利用shRNA壓制內生性Aha1的表現則降低了CLC-1 WT與A531V的表現；這個現象顯示加速Hsp90 ATPase cycling的速率可協助CLC-1的摺疊。此外，我們觀察到大量表現FKBP8後同樣也可增加CLC-1 WT與A531V的穩定性，並且增加了CLC-1在細胞膜上的表現，同時還可減短A531V的半衰期。從上述結果暗示CLC-1很有可能會經由Hsp70-Hsp90進行品質控管，接著我們大量表現Hsc70以及Hsp90α/β，並發現Hsc70以及Hsp90β同樣的增加了CLC-1的穩定性，但Hsp90α則對CLC-1沒有影響。當我們壓制了內生性的Hsc70之後，發現CLC-1 A531V表現量下降。我們也使用了Hsp90的抑制劑17-AAG，並發現CLC-1 WT和A531V的表現皆上升，此現象可能與17-AAG活化了Hsf1並造成其他伴護蛋白增加有關。 從我們的研究結果推測， Hsc70-Hsp90-FKBP8摺疊路徑可能是調控CLC-1蛋白質平衡的關鍵步驟。對於與肌強直症相關的圖變A531V而言，或許經由調控這些相關伴護蛋白的表現可以達成改善其不穩定缺陷的效果。

Myotonia congenita is a hereditary muscle disorder caused by mutations in the human voltage-gated chloride (Cl-) channel CLC-1. A531V is a myotonia-related mutant with a gating property similar to that of wild-type (WT) channels. The protein expression of A531V, however, is significantly lower, which is partly attributed to an enhanced proteasomal degradation. Previous studies from our lab demonstrated that CLC-1 may interact with the Hsp90 cochaperone Aha1, as well as with the PPIase chaperone FKBP8. Molecular chaperones are known to assist protein folding and to remove nonfunctional, misfolded, or aggregated proteins via degradation pathways. In this study, we aim to identify CLC-1-associated molecular chaperones, and to examine whether the chaperones and cochaperones of the Hsp70-Hsp90 system may contribute to the protein quality control of CLC-1 WT and A531V. Overexpression of Aha1 promoted protein expression of both WT and A531V, whereas knockdown of Aha1 reduced the protein expression of both WT and A531V. These observations suggest that the rate of Hsp90 ATPase cycling may be important for the correct folding of CLC-1. Overexpression of FKBP8 also promoted total and surface protein expression of WT and A531V, although only A531V displayed a significant increase in protein half-life. We then investigated the role of Hsp70 and Hsp90 in CLC-1 quality control by overexpressing Hsc70, Hsp90α, or Hsp90β. We found that both Hsc70 and Hsp90β, but not Hsp90α, increased the protein expression of WT and A531V. In addition, knockdown of Hsc70 decreased the expression of A531V. Surprisingly, treatment with 17-AAG, an Hsp90 inhibitor, also led to an increase in the expression of both WT and A531V, which may result from 17-AAG –induced expression of heat shock transcription factor 1 (Hsf1). Together, our data suggest that the Hsc70-Hsp90β-FKBP8 chaperone pathway may play a key role in controlling the protein homeostasis of CLC-1. Our findings also imply that manipulation of the expression/activity of these chaperones/cochaperones may partially correct the protein expression deficit of the myotonia-related mutant A531V.