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系統識別號 U0026-0702201401452500
論文名稱(中文) 亨廷頓相關蛋白40過度表現對於細胞自噬的影響
論文名稱(英文) Effects of HAP40 overexpression on autophagy
校院名稱 成功大學
系所名稱(中) 生命科學系
系所名稱(英) Department of Life Sciences
學年度 102
學期 1
出版年 103
研究生(中文) 黃良卿
研究生(英文) Liang-Ching Huang
學號 l56001020
學位類別 碩士
語文別 中文
論文頁數 82頁
口試委員 指導教授-何盧勳
口試委員-曾淑芬
口試委員-廖泓鈞
中文關鍵字 亨廷頓蛋白  亨廷頓相關蛋白40  細胞自噬  聚集體 
英文關鍵字 Huntingtin  Huntingtin associated protein 40  Autophagy  Aggregate 
學科別分類
中文摘要 亨廷頓舞蹈症為顯性遺傳的神經退化性疾病,致病原因為亨廷頓蛋白基因發生突變,導致突變型亨廷頓蛋白具有過度擴張的多聚麩醯胺,為錯誤折疊的蛋白質易形成聚集體並堆積在神經細胞中。亨廷頓蛋白與多種蛋白質交互作用以參與細胞內的調控,其中亨廷頓蛋白與亨廷頓相關蛋白40結合,可調節早期內噬體的運輸。在患者腦部發現亨廷頓相關蛋白40大量表現,其與微管有交互作用,並與促進突變型亨廷頓蛋白聚集體形成有關。細胞內的清除機制能夠將異常的蛋白質降解以維持細胞恆定,包含泛素-蛋白酶體與細胞自噬,其中泛素-蛋白酶體的組成份子可能被滯留於聚集體,因此細胞自噬的進行顯得十分關鍵,故探討亨廷頓相關蛋白40過度表現對細胞自噬的影響。結果發現亨廷頓相關蛋白40過度表現會促進細胞自噬的進行,進一步發現其作用機制為mTOR-independent,且受到PI-3 kinase的抑制影響。目前對亨廷頓相關蛋白40的研究有限,若能知其在細胞內的分布,將有助於其功能之定位,由於市售HAP40抗體(Millipore AB5872 a.a. 314-325)偵測效果不佳,因此先前製作了兩支HAP40抗體。使用這三種抗體進行特性分析,發現HAP40 (LTK a.a. 293-310)、HAP40 (LTK a.a. 29-47)的偵測較符合HAP40的實際分子量,且knockdown後能偵測到亨廷頓相關蛋白40表現量降低,而市售的HAP40 (Millipore AB5872 a.a. 314-325)抗體則否;另外為了明瞭HAP40分布在細胞核或細胞質,以生化分餾分析,發現亨廷頓相關蛋白40存在於細胞質。在神經細胞中,囊泡藉由運動蛋白在軸突的微管上得以運輸,由驅動蛋白負責正向運輸及動力蛋白負責反向運輸,已知突變型亨廷頓蛋白聚集體內包含運動蛋白,使細胞中游離的運動蛋白減少,影響物質運送。當動力蛋白功能受抑制時,會造成聚集體的堆積,因此想了解驅動蛋白是否也有類似情形。實驗上針對驅動蛋白輕鏈的兩種異構型:KLC1和KLC2進行knockdown,因為輕鏈能夠與物質結合並調控驅動蛋白的活性。結果發現KLC1和KLC2受到抑制時,突變型亨廷頓蛋白聚集體有明顯增加的現象。綜合以上結果顯示亨廷頓相關蛋白40過度表現會促進細胞自噬的進行,其機制為mTOR- independent,且受到PI-3 kinase的抑制所影響;其次分析不同HAP40抗體特性,發現LTK的兩株抗體能夠偵測到內生性HAP40,而由生化分餾結果得知HAP40存在於細胞質;另外KLC1和KLC2表現量下降會導致突變型亨廷頓蛋白聚集體數目增加。
英文摘要 Mutant huntingtin forms aggregates in the brains of transgenic mice and HD patients. Those mutant proteins depend primarily on autophagy for their clearance. Our previous studies showed that HAP40 associates with microtubule and promotes aggregate formation. Therefore, we investigate the effects of HAP40 on autophagy. Using a dual color LC3 reporter to monitor the autophagic process, we found that HAP40 overexpression induced autophagic flux by an mTOR-independent manner. Furthermore, due to the lack of an efficient HAP40 antibody, we have generated and validated two polyclonal HAP40 antibodies. Using those two antibodies, we found that HAP40 signal appears in cytoplasmic fraction. Previous report had shown that depletion of DHC lead to increased aggregate formation. Therefore we investigated whether KLC knockdown have similar effects. We found that there is more aggregates formation after depletion of KLC1 or KLC2.
論文目次 中文摘要 1
Abstract 3
致謝 7
目錄 8
圖目錄 11
縮寫表 12
前言 13
一、亨廷頓舞蹈症 (Huntington's disease, HD) 13
二、亨廷頓蛋白 (Huntingtin, Htt) 13
三、亨廷頓相關蛋白40 (Huntingtin associated protein 40, HAP40) 15
四、亨廷頓蛋白聚集體 (Aggregates) 16
五、驅動蛋白 (Kinesin) 17
六、細胞自噬 (Autophagy) 17
實驗目的 21
材料與方法 22
一、藥品、試劑與質體 22
二、細胞培養 23
三、轉染作用 (Transfection) 27
四、西方墨點法 (Western blot assay) 28
五、即時影像觀察 (Live imaging) 31
六、細胞免疫螢光染色 (Immunofluorescence staining) 31
七、分離萃取細胞核蛋白與細胞質蛋白 32
八、統計方法 (Statistics) 33
結果 34
一、過度表現HAP40對細胞自噬的影響 34
1-1 過度表現HAP40對細胞自噬的影響 34
1-2 抑制溶酶體功能與過度表現HAP40對細胞自噬的影響 36
1-3 抑制HDAC6與過度表現HAP40對細胞自噬的影響 37
1-4 抑制PI 3-kinase與過度表現HAP40對細胞自噬的影響 38
二、HAP40的分布型態 39
2-1 HAP40抗體特性分析 39
2-2 以生化分餾法分析 42
三、確認具專一性辨認KLC的抗體 42
四、降低KLC表現量對亨廷頓蛋白聚集體形成的影響 43
討論 44
參考文獻 49
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