利用Trigger factor以及FKBP12兩種伴護蛋白來調控聚麩胺醯酸蛋白聚集化現象

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李宜妮(YI-NI LI) 查詢紙本館藏

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論文名稱

利用Trigger factor以及FKBP12兩種伴護蛋白來調控聚麩胺醯酸蛋白聚集化現象
(Modulate polyglutamine protein aggregation by Trigger factor and FKBP12)

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摘要(中)

杭丁頓舞蹈症(Huntington’s disease，HD)是一種遺傳性的神經退化疾病，病因由於基因突變造成CAG三核苷酸重複序列過多。三核苷酸CAG所對應的氨基酸為麩胺醯酸(glutamine)，過多的麩胺醯酸片段使得杭丁頓蛋白質產生錯誤摺疊；近來研究指出伴護蛋白可以避免蛋白的錯誤摺疊，抑制因為錯誤 HTT蛋白所造成的神經毒性。根據研究發現在含有過多的麩胺醯酸片段的細胞中過度表達FKBP12伴護蛋白可有效降低細胞死亡率；而另一種伴護蛋白Trigger factor (TF)，其蛋白質中段PPIase區域為FKBP12的同源結構，能與核醣體結合幫助新生蛋白的摺疊。因此，我們利用各種生物物理和生物化學的方法，探討不同的伴護蛋白(TF和FKBP12)是否幫助杭丁頓舞蹈症蛋白質摺疊，改變蛋白質聚集物的含量或性質。
我們成功使用glutathione-S-transferase (GST) 蛋白質系統，表達及純化出不同麩胺醯酸數目的蛋白質(GST-HTT25Q和GST-HTT43Q)。由混濁度測試和filter retardation assay實驗觀察TF和FKBP12對蛋白質聚集物含量上的變化。結果顯示，TF能大幅降低HTT43Q蛋白質聚集體，相反的，FKBP12則會增加HTT43Q蛋白質聚集體含量。穿透式電子顯微鏡觀察蛋白質在TF和FKBP12存在下形態上的變化，HTT43Q在TF存在下培養七天生成HTT43Q單獨在三天時所形成的原纖維結構(protofibril)；HTT43Q在FKBP12存在下一、三和七天都產生大量且沒有特定構形的結構物。然而在Thioflavin T (ThT) 螢光光譜偵測類澱粉纖維含量，TF和FKBP12在一天都有抑制HTT43Q螢光訊號的現象，但七天TF抑制效果下降。同時，拉曼光譜觀測伴護蛋白對二級結構組成比例的差異，分析結果TF和FKBP12的存在皆使得HTT43Q聚集體的二級結構β-sheet比例下降。
我們推論TF和FKBP12伴護蛋白可用兩種截然不同的方式對錯誤摺疊的杭丁頓蛋白質產生作用。TF能延緩聚集化過程，FKBP12則是改變聚集物的構形和性質。此研究幫助我們瞭解伴護蛋白對杭丁頓舞蹈症聚集體的影響，提供未來發展杭丁頓舞蹈症用藥的方向。

摘要(英)

Huntington’s disease (HD) is an autosomal dominant neurodegenerative disease caused by the mutational expansion of CAG triplet repeat in mutant Huntingtin (mHTT) protein. These proteins form aggregates in the affected neurons of patient brains that correlate with disease progression and toxicity. Recent studies reported chaperone can prevent protein misfolding and serve as powerful inhibitor mutant HTT-induced neurotoxicity. According to the recent studies, FKBP12 chaperone had PPIase activity and be decreased the protein level in HD mouse. Another chaperone, Trigger factor (TF), the TFPPIase and FKBP12 are structural homology, can decrease protein aggregates. Here, we investigated whether the presence of FKBP12/TF chaperone could effectively change amounts and properties of polyQ aggregates by biophysical/biochemical technique.
We have successfully established the GST-polyQ system and apply in vitro aggregation assay to unravel the effect of chaperone in aggregation process. Turbidity assay and filter assay revealed TF can significantly suppress HTT43Q aggregates, while FKBP12 increase HTT43Q aggregates. Moreover, we examined the morphology of HTT25Q and HTT43Q in the presence/absence of TF/FKBP12 under Transmission Electron Microscopy (TEM). Massive fibrils can be observed in HTT43Q only. To our surprise, TF significantly changed the morphology of HTT43Q and formed short protofibril structures. Meanwhile, FKBP12 formed amorphous aggregate structures. We further used Thioflavin T (ThT) fluorescence to detect amyloidogenic fibrils. Results showed that TF can FKBP12 can both suppress amyloid fiber at day 1. The inhibition effect can still be seen in FKBP12 but not in TF at day 7, indicating TF can only retard the amyloidogenic process while FKBP12 can shift the process to form non-amyloid aggregates. From the FT-Raman spectroscopy,β-sheet composition both decreased in TF and FKBP12 compared with HTT43Q only.
Here, we propose TF and FKBP12 can modulate mHTT protein aggregation in different pathway. While TF can only retard the amyloidogenic process, FKBP12 can shift the process to form non-amyloid aggregates. This may shed light on the future therapeutical treatments of Huntington’s disease.

關鍵字(中)

★ 杭丁頓
★ 伴護蛋白
★ 蛋白質聚集化

關鍵字(英)

★ huntingtin
★ chaperone
★ protein aggregation

論文目次

中文提要 …………………………………………………………… i
英文提要 …………………………………………………………… ii
誌謝 …………………………………………………………… iii
目錄 …………………………………………………………… iv
圖目錄 …………………………………………………………… v
表目錄 …………………………………………………………… ix
符號說明 …………………………………………………………… x
一、緒論 …………………………………………………………… 1
二、實驗材料與方法…………………………………………………… 11
三、實驗結果…………………………………………………………… 27
四、實驗總結與結果討論……………………………………………… 47
五、參考文獻…………………………………………………………… 52

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指導教授

黃人則、謝發坤
(Joseph Jen-Tse Huang、Fa-Kuen Shieh)

審核日期

2013-6-20

推文