指導教授：陳韻如臺灣大學：生化科學研究所廖宜鴻Liao, Yi-HungYi-HungLiao2014-11-262018-07-062014-11-262018-07-062014http://ntur.lib.ntu.edu.tw//handle/246246/261654類澱粉蛋白斑塊是許多神經退化性疾病的病徵。斑塊的主要成分是乙型類澱粉蛋白，其纖維化目前被接受是造成阿茲海默症的致病原因。然而，乙型類澱粉蛋白的致病性的調控機制至今仍未有全盤的理解，也未有一致的結論。我們在三個章節中探討三個與乙型類澱粉蛋白有關的主題。第一個主題，我們嘗試了解奈米尺度的金粒子是否影響乙型類澱粉蛋白的纖維化過程及形成此影響的原因。實驗結果顯示未經過表面修飾的裸奈米金粒子能以與劑量呈正相關的方式抑制乙型類澱粉蛋白的纖維化，並造成其形成斷裂的類澱粉蛋白纖維與球型多倍體。我們亦發現裸奈米金粒子偏好附著於纖維而非無定型的堆積。此外，奈米金粒子表面的負電位對於其對乙型類澱粉蛋白纖維化的抑制作用有關鍵性的影響。我們的合作人員亦提供細胞毒性測試結果，顯示經過奈米金粒子抑制後的乙型類澱粉蛋白的最終產物對神經細胞的毒性較低。 第二個主題中，我們欲瞭解與家族性遺傳阿茲海默症有關的E22及D23單點突變如何影響同源聚合與異源聚合形成之類澱粉蛋白纖維結構。在我們實驗中，同源聚合指的是單一來源的乙型類澱粉蛋白經培養後形成之纖維，而異源聚合則是混合同濃度之兩種不同乙型類澱粉蛋白 (原生型與突變型) 再經培養後形成的類澱粉蛋白纖維。實驗結果發現同源聚合纖維在蛋白質二級結構上與異源聚合纖維有顯著的不同。而此二集結構上的相異並未反應在類澱粉蛋白纖維螢光探測分子 Thioflavin T與纖維結合的反應計量上。然而，透過ThT與類澱粉蛋白纖維結合後的螢光壽命的測量卻可以得知此參數可能能夠反應纖維結構的異同。我們尚在進行更多相關的實驗以佐證目前實驗結果的初步結論。 我們在最後一個主題探討過渡型金屬離子，特別是二價銅離子與鋅離子，與乙型類澱粉蛋白的交互作用細節及另幾類家族性遺傳相關的 H6及D7單點突變如何影響這交互作用。首先我們提供鋅、銅、鐵、鋁離子與原生型乙型類澱粉蛋白的豪秒級結合動力學，結果顯示這四種金屬離子與乙型類澱粉蛋白結合的速度有顯著差異。接著我們專注於另一類家族性遺傳阿茲海默症變異型H6和D7對於銅離子與乙型類澱粉蛋白結合的影響。實驗結果顯示，除了D7A和H6R外，銅離子結合伴隨著表面疏水性區域的減少。核磁共振實驗結果表明在未加金屬離子前，變異型與原生型乙型類澱粉蛋白在芳香族支鏈光譜區域，特別是H6， H13/H14，和Y10，有些微差異。銅離子結合而導致的光譜消失在D7H變異型中較為不顯著。1H-15N異核核磁共振光譜顯示銅離子與D7H和原生型乙型類澱粉蛋白結合的相異之處。Amyloid plagues are the major pathogenic hallmarks in many neurodegenerative diseases. Fibrillization of the primary constituents, amyloid-β (Aβ), in plagues is considered the pathogenesis of Alzheimer’s disease (AD). Conclusive evidence is still lacking to reach a comprehensive and convergent view of how Aβ is modulated in the pathogenesis of AD. Here, in three separate chapters, we examined how gold nanoparticles (AuNPs) interfere with fibrillization of Aβ, the effects of single-point N-terminal mutation, H6 and D7, on the interactions between metal ions and Aβ, and how single-point mutations, E22 and D23, influence the structures of fibril. In Chapter 3, experimental results showed that (1) bare AuNPs inhibited Aβ fibrillization in a dose-dependent manner and redirected Aβ forming fragmented fibrils and spherical oligomers; (2) bare AuNPs bound preferentially to Aβ fibrils but not amorphous aggregates; (3) negative surface potential of AuNPs was required for inhibitive effect. In conjunction with our in vitro biophysical and biochemical data, cell studies from our lab member revealed that in our experimental settings, inhibition of Aβ fibrillization by negatively charged AuNPs also reduce Aβ-induced cytotoxicity in neuronal cells. In Chapter 4, we aimed to reveal distinctions among structures of homopolymeric fibrils (homo-fibrils) formed from incubation of monomers of a single species, and heteropolymeric ones (hetero-fibrils) started from incubation of equal-molar-mixed monomers of wild type and an Aβ variant. We showed that E22 hetero-fibrils are distinct from E22 homo-fibrils in terms of secondary structures. Perturbed secondary structure could influence conformational packing of cross β-sheet, which were not revealed by stoichiometry between Aβ and ThT but by ThT fluorescence lifetime. In Chapter 5, we investigated millisecond binding kinetics of four metal ions, Zn2+, Cu2+, Fe3+, and Al3+. We then focused on Cu2+ and demonstrated that mutation at residues H6 and D7 impact on Cu2+ binding affinities. We found by Bis-ANS fluorescence that diminished hydrophobic exposure was accompanied by Cu2+ binding except for D7A and H6R. Our preliminary proton NMR showed that there existed environmental variance surrounding residues H6, H13/H14, and Y10 among monomeric Aβ variants in native condition. Cu2+-binding-induced peak loss was less evident in D7 mutants. 1H-15N NMR HSQC spectroscopy provided additional information regarding the residual involvement on Cu2+ binding between D7H and wild type Aβ.Table of Contents 口試委員會審定書 .i 誌謝........... ii 中文摘要...... iv Abstract...... vi Abbreviations viii Table of Contents x List of Figures xiv List of Tables xviii Chapter 1 Introduction 1 1.1 Alzheimer’s disease 1 1.1.1 Overview 1 1.1.2 Risk factors and treatments 3 1.1.3 Hypotheses of the pathogenesis of Alzheimer’s Disease 4 1.2 Amyloid-β 5 1.2.1 Amyloid-β monomer 8 1.2.2 Amyloid-β fibrils 9 1.3 Amyloid-β and metal ions 11 1.4 Single-point mutations of Amyloid-β 13 1.4.1 Familial mutations, A21, E22, and D23, in Alzheimer’s disease 13 1.4.2 N-terminal familial mutations, H6 and D7, in Alzheimer’s disease 15 1.5 Application of nanotechnology in the studies of Alzheimer’s disease 16 Chapter 2 Materials and methods 18 2.1 Materials 18 2.2 Methods 18 2.2.1 Aβ monomer preparation 18 2.2.2 AuNPs preparation 20 2.2.3 Thioflavin T assay 21 2.2.4 Dynamic light scattering 21 2.2.5 Transmission electron microscopy 22 2.2.6 Fourier transform infrared spectroscopy 22 2.2.7 Far-UV circular dichroism spectroscopy 23 2.2.8 Fluorescence lifetime measurement 23 2.2.9 Stopped-flow fluorescence 24 2.2.10 Fluorescence spectroscopy 25 2.2.11 Proton and 1H-15N HSQC NMR spectroscopy 25 Chapter 3 Interference of gold nanoparticles in the fibrillization process of wild type Aβ40.......... 27 3.1 Aims 27 3.2 Bare AuNPs inhibit Aβ fibrillization without self-clustering 28 3.3 Bare AuNPs alter ThT fluorescence intensity of preformed Aβ fibrils 31 3.4 Absorbance spectra of Aβ monomers or preformed Aβ fibrils 33 3.5 DLS analysis shows redirected Aβ species in the presence of AuNPs 35 3.6 Bare AuNPs induce Aβ fibril fragmentation and oligomer formation and preferentially bind to Aβ fibrils 37 3.7 Negatively charged AuNPs inhibit Aβ fibrillization in vitro 40 Chapter 4 Structural differences between homo-polymeric and hetero-polymeric Aβ fibrils.......... 43 4.1 Aims 43 4.2 Homo- and hetero- fibrils are morphologically indistinguishable under TEM imaging. 44 4.3 Binding stoichiometry of Thioflavin T to amyloid-β fibrils 46 4.4 Secondary structures of fibrils revealed by far-UV circular dichroism 48 4.5 Secondary structures of fibrils revealed by Fourier transformation infrared spectroscopy 52 4.6 Fluorescence lifetime of bound ThT on fibrils 57 Chapter 5 Interactions of Cu2+ with wild type and N-terminal familial mutants of Aβ 62 5.1 Aims 62 5.2 Metal ion binding examined by stopped-flow fluorescence. 62 5.3 Intrinsic fluorescence of H6 and D7 mutants upon Cu2+ binding 65 5.4 Bis-ANS fluorescence of H6 and D7 mutants upon Cu2+ binding 68 5.5 1H NMR spectroscopy 72 5.6 1H-15N HSQC spectroscopy of D7H and wild type Aβ 78 Chapter 6 Discussion 84 6.1 Negatively Charged Gold Nanoparticles Inhibit Alzheimer’s Amyloid-β Fibrillization, Induce Fibril Dissociation, and Mitigate Neurotoxicity 84 6.1.1 Results of various effects imposed by AuNPs on Aβ 84 6.1.2 Inhibition of Aβ fibrillization by nanoparticles 87 6.1.3 Effect of localized charge on Aβ fibrillization 88 6.2 Homo- and hetero- polymeric Aβ fibrils 91 6.3 Cu2+ coordination of H6 and D7 mutants of Aβ 92 6.3.1 Effect of H6 mutation on Cu2+ binding to Aβ 94 6.3.2 Effect of D7 mutation on Cu2+ binding to Aβ 95 Reference... 97 Supplementary data 1084324030 bytesapplication/pdf論文公開時間：2014/08/17論文使用權限：同意無償授權阿茲海默症乙型類澱粉蛋白類澱粉蛋白纖維家族性突變型金屬離子奈米金粒子thesishttp://ntur.lib.ntu.edu.tw/bitstream/246246/261654/1/ntu-103-D96B46015-1.pdf