博碩士論文 102223010 詳細資訊




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姓名 楊耀甄(Yao-chen Yang)  查詢紙本館藏   畢業系所 化學學系
論文名稱 伴護蛋白藉由抑制異常亨丁頓蛋白聚集化與寡聚化現象以引發良性效應
(The suppression of aggregation/oligomerization process of mHTT protein by chaperones elicits beneficial effect)
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摘要(中) 亨丁頓氏舞蹈症(Huntington’s disease, HD)是由於亨丁頓蛋白(Huntingtin)含有過多的麩醯胺酸(glutamine, Gln, Q)導致其聚集化形成大量的包涵體(inclusion body)累積。根據研究指出分子伴護蛋白除了一般認知上的幫助多肽鏈的摺疊,也會清除錯誤摺疊的蛋白質避免聚集化。其中,與核醣體相關的伴護蛋白──Trigger Factor(TF),在原核生物界負責幫助大部分的新生成多肽鏈之摺疊。在本篇研究中我們利用生物物理化學的方法證明了TF在in vitro及in vivo的環境中確實會影響異常亨丁頓蛋白(mutant huntingtin, mHTT)的聚集化。首先我們運用GST-HTT polyQ蛋白系統探討TF對聚集化過程的影響,另外也以研究最為廣泛的DnaK伴護蛋白一起比較。由濾膜滯留分析法結果顯示TF及DnaK會使得mHTT的聚集物大量減少。透過穿透式電子顯微鏡觀察,mHTT的聚集化過程在TF與DnaK的存在下有所減緩。接著我們更進一步的以細胞實驗探討TF的作用。引人注目的是,於TF的存在下,mHTT的聚集物與寡聚物皆有減少的現象,且TF對神經突分化與細胞存活率有增加的作用。綜合實驗結果,我們發現TF的新作用──調控mHTT的聚集化及寡聚化過程,提供了對亨丁頓氏舞蹈症的治療方向。
摘要(英) The abundant accumulation of inclusion bodies containing polyglutamine (polyQ)-expanded mutant huntingtin (mHTT) aggregates is considered as the key pathological event in Huntington’s disease (HD). Literatures have reported that molecular chaperones participate in multiple cellular processes including assisting the folding of newly translated/damaged polypeptides and clearance of the misfolded proteins. Here, we show that Trigger factor (TF), a ribosome-associated chaperone responsible for facilitating the folding of nascent polypeptides in prokaryotes, is able to impact mHTT aggregation in vitro and in vivo.First, we applied GST-HTT polyQ protein system to explore the influence of TF and DnaK, a classical chaperone, in the aggregation process. Our result showed both TF and DnaK significantly reduced HTT(Q)43 aggregates as examined by filter retardation assay. Through transmission electron microscopy (TEM) observation, the mHTT aggregation process was retarded in the presence of TF similar as DnaK. We further examined the biological role of TF in mHTT-expressing N2A cells. Strikingly, we discovered mHTT oligomers were decreased in presence of TF. Furthermore, TF showed increased cell viability and neurite outgrowth in mHTT-expressing cells. Taken these together, we discovered a novel function of TF in modulating the oligomerization/aggregation process, which may benefit developments in HD therapeutic strategies in the future.
關鍵字(中) ★ 亨丁頓氏舞蹈症
★ 亨丁頓蛋白
★ 蛋白質聚集化
★ 蛋白質寡聚化
★ 伴護蛋白
關鍵字(英)
論文目次 中文摘要 i
Abstract ii
誌 謝 iii
目 錄 v
圖目錄 vii
表目錄 ix
符號說明 x
第一章 緒 論 1
1-1 亨丁頓氏舞蹈症(HD) 1
1-2 亨丁頓蛋白質(huntingtin, HTT) 2
1-3 聚麩醯胺酸(polyglutamine)蛋白質之錯誤摺疊 3
1-3-1 錯誤摺疊的蛋白質聚集化 4
1-3-2 寡聚化過程 4
1-4 分子伴護蛋白(molecule chaperones)的角色 5
1-4-1 Trigger Factor (TF)伴護蛋白 6
1-4-2 DnaK伴護蛋白 7
1-5 研究目的 8
1-6 實驗流程 9
第二章 實驗材料與方法 10
2-1 實驗材料與儀器 10
2-1-1 實驗藥品 10
2-1-2 實驗緩衝溶液 11
2-1-3 實驗儀器 12
2-2 蛋白質的表達與純化 12
2-2-1 GST fusion protein 13
2-2-2 DnaK的表達與純化 13
2-3 西方點墨法(western blot) 14
2-4 GST-HTT polyQ蛋白質聚集化測試 15
2-5 濾膜滯留分析法(Filter retardation assay) 15
2-6 穿透式電子顯微鏡影像(Transmission Electron Microscopy) 16
2-7 細胞培養和表達mHTT與其聚集化作用 17
2-8 細胞內mHTT的寡聚化作用 17
2-8-1 狹缝雜交法(slot-blot assay) 18
2-8-2 西方點墨法 18
2-9 共軛焦顯微鏡影像(Confocal microscopy) 18
2-10 良性效應(beneficial effect) 19
第三章 實驗結果 21
3-1 蛋白質表達與純化 21
3-1-1 DnaK的表達與純化 21
3-1-2 蛋白質的製備(GST-HTT(Q)43、GST-HTT(Q)25、TF、DnaK) 22
3-2 GST-HTT(Q)43蛋白質的聚集化測試 23
3-3 濾膜滯留分析法(Filter retardation assay) 24
3-3-1 TF對HTT(Q)43蛋白質聚集化的影響 24
3-3-2 TF的劑量多寡對HTT(Q)43蛋白質聚集化的影響 26
3-4 穿透式電子顯微鏡影像觀察HTT (Q)43蛋白質的形態 27
3-5 利用細胞表達蛋白質, 檢測TF對HTT polyQ蛋白質聚集化的影響 29
3-5-1 在細胞中TF對HTT polyQ蛋白聚集化的影響 29
3-5-2 在細胞中TF的劑量對Htt (Q)109-eYFP蛋白質聚集化的作用 29
3-6 利用細胞表達蛋白質, 檢測TF對HTT polyQ蛋白質寡聚化的影響 30
3-7 共軛焦顯微鏡影像觀察HTT-polyQ與TF的分布 33
3-8 TF對於神經突分化及細胞存活率的影響 34
第四章 總結與討論 36
4-1 實驗總結 36
4-2 結果討論 38
參 考 文 獻 40
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指導教授 黃人則、謝發坤(Jen-Tse Huang Fa-Kuen Shieh) 審核日期 2015-7-20
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