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研究生:陳容碩
研究生(外文):Rong-Shuo Chen
論文名稱:研究阿茲海默症中TDP-43與乙型類澱粉胜肽之間的分子交互作用
論文名稱(外文):Understanding the Molecular Interaction of TDP-43 and Amyloid-β in Alzheimer’s disease
指導教授:陳韻如陳韻如引用關係
指導教授(外文):Yun-Ru Chen
口試委員:楊健志廖憶純杜玲嫻
口試委員(外文):Chien-Chih YangYi-Chun LiaoLing-Hsien Tu
口試日期:2019-07-18
學位類別:碩士
校院名稱:國立臺灣大學
系所名稱:生化科技學系
學門:生命科學學門
學類:生物科技學類
論文種類:學術論文
論文出版年:2019
畢業學年度:107
語文別:英文
論文頁數:65
中文關鍵詞:阿茲海默症乙型類澱粉胜肽TDP-43蛋白質間交互作用抗TDP-43寡聚體的單株抗體
DOI:10.6342/NTU201903526
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漸凍人症的病理特徵,TDP-43沉積,在阿茲海默症的病人中相當常見,這代表著TDP-43可能在阿茲海默症中是除了兩個為人所知的病理特徵乙型類澱粉蛋白 (Aβ)以及tau蛋白之外,第三個重要的病理特徵。根據陳韻如老師實驗室過去的研究結果,TDP-43可以影響Aβ40的堆疊、使之保持在寡聚體的階段、並證實兩者間有交互作用,這些研究結果促使我想去探討TDP-43與Aβ40之間的分子交互作用。
首先,在Thioflavin-T檢測中,藉由讓特定幾個抗TDP-43寡聚體的單株抗體或市售的抗TDP-43的單株抗體與TDP-43結合,TDP-43抑制Aβ40聚集的效能大幅降低,因此我發現TDP-43藉由其N端或是透過第二個與核糖核酸結合的位置(RRM2)和Aβ40產生交互作用。此外,雖然小鼠和人類的TDP-43主要差異在RRM2的序列,兩種蛋白都會影響Aβ40的堆疊。第二,在蛋白質核磁共振實驗中,我發現準備Aβ40所使用的緩衝液會造成TDP-43蛋白質構型改變,並可能進而使TDP-43無法和Aβ40有交互作用。但在改變Aβ40的準備方式後,我們仍發現氮15同位素標定的TDP-43 N端核磁共振圖譜並沒有發生改變,顯示在此條件下兩者無穩定作用。第三,在酵素免疫分析法中,藉由讓特定幾個抗TDP-43寡聚體的自製單株抗體或市售的抗TDP-43的單株抗體先與TDP-43結合,biotin-Aβ40就無法或降低與TDP-43的結合,因此我發現TDP-43藉由其C端以及RRM1+2與biotin-Aβ40有交互作用。由此我們推測,Aβ40與TDP-43有著多個交互作用的位置,確切位置仍需後續證明。
ALS disease hallmarks, TDP-43 pathology, were rather common in Alzheimer’s disease (AD), which suggested that TDP-43 might be the third important disease hallmarks in AD besides the well-known AD hallmarks, amyloid-β (Aβ) and tau protein. From the previous results of Dr. Yun-Ru Chen’s lab, we found that TDP-43 could affect Aβ40 aggregation, retain it in its oligomeric state, and interact with it, which motivated me to investigate how did they interact in molecular level.
Firstly, in Thioflavin-T assay we demonstrated TDP-43 might interact with Aβ40 by its N-terminal or RNA recognition motif 2 (RRM2) region with the aid of selected self-prepared TDP-O antibodies and commercial antibodies. In addition, both mouse and human TDP-43 could affect Aβ40 aggregation although mouse TDP-43 mainly differed with human TDP-43 in RRM2 motif. Secondly, in 1H-15N HSQC protein NMR experiments we found that 15N-Aβ40 preparation buffer altered TDP-43 conformation and might interrupt the interaction between 15N-Aβ40 and TDP-43. But we still demonstrated no significant changes in N-terminal of 15N-TDP-43 NMR graph after Aβ40 preparation was changed. The results showed that in the experimental condition, the two proteins did not stably interact. Thirdly, in ELISA assay, we demonstrated TDP-43 interacted with biotin-Aβ40 by its C-terminal or RRM1+2 domain with the aid of our TDP-O antibodies and the commercial antibodies. Our results suggested that Aβ40 could interact with multiple domains of TDP-43 and the specific interaction sites need further examination.
誌謝 i
中文摘要 ii
Abstract iii
Contents iv
List of Figures vi
Chapter 1 Introduction: 1
1.1 Dementia: 1
1.2 Alzheimer’s disease 1
1.2.1 The discovery of Alzheimer’s disease 1
1.2.2 Main hypothesis in AD and drug development 2
1.2.3 Aβ production and the structural characteristics 3
1.3 TAR DNA-binding protein 43 (TDP-43) 4
1.3.1 The discovery of TDP-43 4
1.3.2 The physiological function of TDP-43 5
1.3.3 The discovery and importance of TDP-43 in Alzheimer’s disease 6
1.3.4 The generation of monoclonal TDP-O antibodies 6
1.3.5 Possible extracellular and intracellular interaction between TDP-43 and Aβ40 7
1.4 Aim of my research 8
Chapter 2 Material and methods 10
2.1 Protein expression and purification 10
2.1.1 TDP-43 (transactive response DNA binding protein 43 kDa) 10
2.1.2 Aβ40 expression and purification 13
2.2 dot blot 16
2.3 Size exclusion chromatography 17
2.4 Western blot 17
2.5 Thioflavin T assay 18
2.6 1H-15N Heteronuclear Single-Quantum Coherence (HSQC) nuclear magnetic resonance of protein (protein NMR) 19
2.6.1 15N labeled Aβ40 19
2.6.2 15N labeled TDP-43 (1-100) 20
2.7 ELISA 21
Chapter 3 Results 23
3.1 Protein purification 23
3.1.1 Full-length and truncated human TDP-43 23
3.1.2 Aβ40 purification 26
3.1.3 Characterization of monoclonal anti-human TDP-43 oligomer antibodies 26
3.2 Examination of interactions between TDP-43 and Aβ40 28
3.2.1 ThT assay 29
3.2.2 1H-15N Heteronuclear Single-Quantum Coherence nuclear magnetic resonance of protein (1H-15N HSQC protein NMR) 31
3.2.3 Enzyme-linked immunosorbent assay (ELISA) 34
Chapter 4. Discussion 37
Figures 41
References: 62
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