臺灣博碩士論文加值系統

普立昂疾病是一類具傳染力且致命的神經退化性疾病，會侵襲中樞神經系統造成人類或動物腦組織產生海綿狀的病變。其致病物質為錯誤折疊的普立昂蛋白(PrPSc)，有容易聚集和傳染的特性。細胞中正常的普立昂蛋白(PrPC)是α螺旋為主的結構，會轉變成以β摺板為主的致病性普立昂蛋白(PrPSc)，而聚集成易沉澱的澱粉樣類纖維構造。這種不正常的結構轉變便是造成細胞毒性的主要原因。但是目前我們仍然不清楚普立昂疾病結構轉變的過程和致病機制。本實驗室在先前研究中發現雙硫鍵被移除的重組普立昂蛋白，可以在中性環境下進行自發性的結構轉變。它可以轉變成β寡聚體或者澱粉樣類纖維構造。為了探討不同區域的資訊，在這項研究中，我們先分別標定在普立昂蛋白的三個α螺旋上。接著，將標定的普立昂蛋白進行結構轉變，包括自發性結構轉變，形成β寡聚體以及形成澱粉樣類纖維構造。我們利用圓二色光譜和電子自旋共振(ESR)圖譜等技術來探討這些區域有無參與結構轉變。在自發性的結構轉變中，我們發現整體結構變得比較鬆散。另外，在β寡聚體的結構時，helix 1和helix 2的結構會部分解開，而helix 3可能維持在穩定的結構中。最後，在澱粉樣類纖維構造中，我們發現helix 3的分子間距離只有1奈米，但仍然需要進一步實驗來確認β摺板為主結構是由哪一區域轉變成的。

Transmissible Spongiform Encephalopathies (TSE), also called prion diseases are infectious neurodegenerative disorders. The key molecular event in the pathogenesis of prion diseases is the conformational conversion of a cellular prion protein, PrPC, into a misfolded form, PrPSc. The α to β conformational transition leads to protein aggregation and the formation of toxic amyloid fibrils. However, the mechanism of protein misfolding and the pathogenic pathway are still unclear. In our lab’s previous study, we found that the disulfide-deleted mouse PrP could undergo a spontaneous structural conversion under native condition from the native α-helical structure to β-oligomers, amorphous aggregate, even amyloid fibrils. Here, we made spin-labeling on each of three helices of mouse prion protein individually and combined circular dichroism spectroscopy and electron spin resonance (ESR) spectroscopy to investigate the structural conversion process. Our study illustrated that helix 1 and helix 2 were partially unfolded when converted into soluble β-structures. On the other hand, spin labeled on helix 3 showed slow mobility, suggesting that the local environment of that spin is in an ordered state. Moreover, when the helix3-labeled protein was transformed into amyloid fibrils, the spin-labeled fibrils showed intermolecular spin interaction with a distance of 10 A.

中文摘要 III
Abstract IV
Abbreviations V
Contents VIII
Figure contents XI
Chapter 1 Introduction 1
1.1 Introduction to prion disease 1
1.2 Protein only hypothesis 5
1.3 Prion infectious conversion 6
1.4 The structural and biological aspect of PrPC 9
1.5 Different models of PrPSc 11
1.6 Electron spin resonance (ESR) 19
1.7 Previous study in our lab 24
1.8 The aim of the thesis 25
Chapter 2 Materials and Methods 26
2.1 Materials 26
2.1.1 Water 26
2.1.2 Chemicals 26
2.2 Methods 29
2.2.1 Expression construct and site-directed mutagenesis 29
2.2.2 Small-scale protein expression 30
2.2.3 Large-scale protein expression, purification, and identification 31
2.2.3.1 Glycerol cell stock preparation 31
2.2.3.2 Expression of recombinant mouse PrP in E. coli and cell lysis 31
2.2.3.3 Immobilized metal-ion affinity chromatography (IMAC) 32
2.2.3.4 Desalting and disulfide bond formation for mPrPwt 33
2.2.3.5 HPLC purification and protein identification 33
2.2.4 Secondary structure analysis by circular dichroism and CDPro 35
2.2.5 Analytical ultracentrifugation (AUC) 35
2.2.6 Fibril formation and ThT (thioflavin T) binding assay 36
2.2.7 Transition electron microscopy (TEM) 37
2.2.8 Spin-labeling & purification 38
2.2.9 Electron spin resonance (ESR) 39
2.2.10 Pepsin digestion assay 40
Chapter 3 Results (I) 41
3.1 Design and expression of mutant mouse prion protein constructs 41
3.2 Small-scale expression analysis 43
3.3 large-scale expression 44
3.4 Purification by immobilized metal-ion affinity chromatography (IMAC) 45
3.5 Desalting and disulfide bond formation for mPrPwt 46
3.6 High-performance liquid chromatography (HPLC) purification 47
3.7 Spin-labeling and purification 49
3.8 Protein identification and storage 52
Chapter 4 Results (II) 57
4.1 Spontaneous structural conversion under native condition 57
4.1.1 CD spectra of spontaneous structural conversion 57
4.1.2 Monitor the spontaneous structural conversion by ESR 60
4.1.3 pH value can affect structural conversion rate 64
4.2 Soluble β-PrP 68
4.2.1 CD spectra of β-PrP 68
4.2.2 Particle size determination 69
4.2.3 TEM image and ThT assay of β-PrP 70
4.2.4 ESR spectra of β-PrP 73
4.3 Amyloid fibrils 75
4.3.1 ThT binding assay and TEM images of mutant PrP 75
4.3.2 Fibrils formed from Spin-labeled PrP 78
4.3.3 ESR spectra of amyloid fibrils 80
4.3.4 Pepsin digestion assay 81
Chapter 5 Discussion 84
Chapter 6 Future Works 90
References 91

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