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| DC 欄位 | 值 | 語言 |
|---|---|---|
| dc.contributor.advisor | 陳佩燁 | zh_TW |
| dc.contributor.advisor | Rita Pei-Yeh Chen | en |
| dc.contributor.author | 顏歆恩 | zh_TW |
| dc.contributor.author | Hsin-En Yen | en |
| dc.date.accessioned | 2025-09-10T16:23:38Z | - |
| dc.date.available | 2025-09-11 | - |
| dc.date.copyright | 2025-09-10 | - |
| dc.date.issued | 2025 | - |
| dc.date.submitted | 2025-07-30 | - |
| dc.identifier.citation | 1. Prusiner SB: Prions. Proceedings of the National Academy of Sciences of the United States of America 1998, 95(23):13363-13383.
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Forloni G, Angeretti N, Chiesa R, Monzani E, Salmona M, Bugiani O, Tagliavini F: Neurotoxicity of a prion protein fragment. Nature 1993, 362(6420):543-546. 25. Brown DR, Herms J, Kretzschmar HA: Mouse cortical cells lacking cellular PrP survive in culture with a neurotoxic PrP fragment. Neuroreport 1994, 5(16):2057-2060. 26. Brown DR, Schmidt B, Kretzschmar HA: Role of microglia and host prion protein in neurotoxicity of a prion protein fragment. Nature 1996, 380(6572):345-347. 27. Tagliavini F, Prelli F, Verga L, Giaccone G, Sarma R, Gorevic P, Ghetti B, Passerini F, Ghibaudi E, Forloni G et al: Synthetic peptides homologous to prion protein residues 106-147 form amyloid-like fibrils in vitro. Proceedings of the National Academy of Sciences of the United States of America 1993, 90(20):9678-9682. 28. Norstrom EM, Mastrianni JA: The AGAAAAGA palindrome in PrP is required to generate a productive PrPSc-PrPC complex that leads to prion propagation. Journal of Biological Chemistry 2005, 280(29):27236-27243. 29. Mastrianni JA: The genetics of prion diseases. Genetics in Medicine 2010, 12(4):187-195. 30. Jeong BH, Kim YS: Genetic studies in human prion diseases. Journal of Korean Medicine Sci 2014, 29(5):623-632. 31. Stahl N, Borchelt DR, Hsiao K, Prusiner SB: Scrapie prion protein contains a phosphatidylinositol glycolipid. Cell 1987, 51(2):229-240. 32. Zhao H, Wang XY, Zou W, Zhang YP: Prion protein gene (PRNP) polymorphisms in native Chinese cattle. Genome 2010, 53(2):138-145. 33. Billeter M, Riek R, Wider G, Hornemann S, Glockshuber R, Wüthrich K: Prion protein NMR structure and species barrier for prion diseases. Proceedings of the National Academy of Sciences of the United States of America 1997, 94(14):7281-7285. 34. Vassar PS, Culling CF: Fluorescent stains, with special reference to amyloid and connective tissues. Archives of Pathology & Laboratory Medicine 1959, 68:487-498. 35. Biancalana M, Koide S: Molecular mechanism of Thioflavin-T binding to amyloid fibrils. Biochimica et Biophysica Acta 2010, 1804(7):1405-1412. 36. Scheres SH: RELION: implementation of a Bayesian approach to cryo-EM structure determination. Journal of Structural Biology 2012, 180(3):519-530. 37. Chen EH, Kao HW, Lee CH, Huang JYC, Wu KP, Chen RP: 2.2 Å Cryo-EM Tetra-Protofilament Structure of the Hamster Prion 108-144 Fibril Reveals an Ordered Water Channel in the Center. Journal of the American Chemical Society 2022, 144(30):13888-13894. 38. Matsuo K, Hiramatsu H, Gekko K, Namatame H, Taniguchi M, Woody RW: Characterization of intermolecular structure of β(2)-microglobulin core fragments in amyloid fibrils by vacuum-ultraviolet circular dichroism spectroscopy and circular dichroism theory. Journal of Physical Chemistry B 2014, 118(11):2785-2795. 39. Wadsworth JD, Asante EA, Desbruslais M, Linehan JM, Joiner S, Gowland I, Welch J, Stone L, Lloyd SE, Hill AF et al: Human prion protein with valine 129 prevents expression of variant CJD phenotype. Science 2004, 306(5702):1793-1796. 40. Chen VB, Arendall WB, 3rd, Headd JJ, Keedy DA, Immormino RM, Kapral GJ, Murray LW, Richardson JS, Richardson DC: MolProbity: all-atom structure validation for macromolecular crystallography. Acta Crystallogr D Biol Crystallogr 2010, 66(Pt 1):12-21. 41. Eraña H, Venegas V, Moreno J, Castilla J: Prion-like disorders and Transmissible Spongiform Encephalopathies: An overview of the mechanistic features that are shared by the various disease-related misfolded proteins. Biochemical and Biophysical Research Communications 2017, 483(4):1125-1136. 42. Goedert M: NEURODEGENERATION. Alzheimer's and Parkinson's diseases: The prion concept in relation to assembled Aβ, tau, and α-synuclein. Science 2015, 349(6248):1255555. 43. Hallinan GI, Ozcan KA, Hoq MR, Cracco L, Vago FS, Bharath SR, Li D, Jacobsen M, Doud EH, Mosley AL et al: Cryo-EM structures of prion protein filaments from Gerstmann-Sträussler-Scheinker disease. Acta Neuropathologica 2022, 144(3):509-520. 44. Glynn C, Sawaya MR, Ge P, Gallagher-Jones M, Short CW, Bowman R, Apostol M, Zhou ZH, Eisenberg DS, Rodriguez JA: Cryo-EM structure of a human prion fibril with a hydrophobic, protease-resistant core. Nature Structural & Molecular Biology 2020, 27(5):417-423. 45. Zhang X, Xu H, Tang H, Lv Z, Zou Y, Huang F, Ding F, Sun Y: The Glycine-Rich Region as a Flexible Molecular Glue Promoting hPrP(106-145) Aggregation into β-Sheet Structures. Journal of Chemical Information and Modeling 2025. | - |
| dc.identifier.uri | http://tdr.lib.ntu.edu.tw/jspui/handle/123456789/99472 | - |
| dc.description.abstract | 普利昂疾病(Prion disease)是一種可由錯誤摺疊的蛋白質傳染的致死性神經退化性疾病，並可在好幾種哺乳類動物之間跨種傳染，其致病機制源於正常細胞膜上的普利昂蛋白PrPC (cellular prion protein)轉變為錯誤摺疊的PrPSc (scrapie prion protein)後，堆疊形成富含β-sheet的類澱粉纖維並在腦部累積，最後造成腦細胞死亡並在腦中留下很多海綿狀的空洞。
在不同物種間的跨種傳染存在著因序列差異所導致的物種感染屏障(sequence-dependent transmission barrier)，例如牛會感染貓和人，卻不會感染狗和豬。人的普利昂蛋白的第129號胺基酸有多型性，可以是methionine (M) 或是valine (V)，根據截至目前為止之流行病學報告，因狂牛症而感染新型庫賈氏症(new variant Creutzfeldt-Jakob disease，v-CJD)的患者中只有一名是129M/V異型接合子, 其他新型庫賈氏症患者全都是129M同型接合子。 本研究著重探討胺基酸序列與類澱粉纖維結構及引晶效率的關係，由於普利昂蛋白的危險等級為3，因此我們僅研究沒有生物體感染性，但對於普利昂纖維形成很重要的序列108-144，分別合成牛、貓、及人類(129M與129V)等物種之普利昂胜肽去形成類澱粉纖維。研究結果顯示四種普利昂胜肽纖維展現了具差異性的Thioflavin T螢光強度與親和性，依此特性進一步將其分成三個組別；交叉引晶(cross-seeding)方法表明人類129V普利昂胜肽(huPrP(108-144)129V)有較高的引晶障礙(seeding barrier)並只有人類129M普利昂胜肽(huPrP(108-144)129M)有能力感染huPrP(108-144)129V。另一方面，我們亦利用冷凍電子顯微鏡去進行所形成的牛普利昂胜肽(bPrP(108-144))類澱粉纖維的結構分析。 | zh_TW |
| dc.description.abstract | Prion disease is a fatal neurodegenerative disease that can be transmitted between multiple mammalian species through misfolded proteins. The pathogenic mechanism involves the conversion of normal protein PrPC (cellular prion protein) into pathological isoform PrPSc (scrapie prion protein), which undergoes self-propagating misfolding and accumulates as β-sheet-rich amyloid-like fibrils in neural tissues. This accumulation ultimately leads to neuronal death and sponge-like cavities in the brain.
Interspecies transmission is caused by sequence-dependent infection barriers, such as the ability of cattle to infect cats and humans but not dogs and pigs. Human prion proteins exhibit polymorphism at the 129th amino acid position, either methionine (M) or valine (V). To date, epidemiological data indicate that among those infected with variant Creutzfeldt-Jakob disease (v-CJD) from mad cow disease, all patients were 129M homozygotes except for one 129M/V heterozygote. This research focuses on investigating the relationship between amino acid sequences, amyloid-like fibril structures, and seeding efficiency. The study concentrates on the non-infectious sequence 108-144, which is crucial for fibril formation. Synthesized prion peptides from bovine, cat, and human (129M and 129V) were used for analysis. The study results identified varying levels of Thioflavin T fluorescence intensity and binding profile across four different prion peptide fibers, leading to their classification into three groups. Cross-seeding studies revealed that huPrP(108-144)129V exhibits a higher seeding barrier, with only the huPrP(108-144)129M capable of inducing fibril formation in huPrP(108-144)129V. Additionally, cryogenic electron microscopy (cryo-EM) was employed to resolve the fibril structure of bPrP(108-144). | en |
| dc.description.provenance | Submitted by admin ntu (admin@lib.ntu.edu.tw) on 2025-09-10T16:23:38Z No. of bitstreams: 0 | en |
| dc.description.provenance | Made available in DSpace on 2025-09-10T16:23:38Z (GMT). No. of bitstreams: 0 | en |
| dc.description.tableofcontents | 致謝 i
摘要 iii Abstract iv Abbreviations v Contents vii List of figures xi List of tables xv List of appendices xvi Chapter 1 Introduction 1 1.1 Prion disease 1 1.2 Species transmission barrier 2 1.3 Prion peptide 108-144 6 1.4 Aim of this thesis 9 Chapter 2 Materials and Methods 11 2.1 Materials 11 2.1.1 Water 11 2.1.2 Chemicals 11 2.1.3 Instruments 12 2.2 Methods 13 2.2.1 Expression of Prion Protein 108-144 Peptide Segment 13 2.2.2 Amyloid Fibril Formation 16 2.2.3 ThT Binding Assay 16 2.2.4 Cross-seeding Assay 18 2.2.5 Circular Dichroism (CD) Spectroscopy 19 2.2.6 Negative-Stain Transmission Electron Microscopy (TEM) 20 2.2.7 Atomic Force Microscope (AFM) 20 2.2.8 Cryogenic Electron Microscopy (cryo-EM) 21 Chapter 3 Results Ⅰ. Prion peptides preparation 23 3.1 Synthesis and purification of four prion peptides 23 Chapter 4 Results Ⅱ. Structure conversion of four prion peptides 27 4.1 Fiber formation assay of bPrP(108-144), cPrP(108-144), huPrP(108-144)129M, and huPrP(108-144)129V 27 4.2 TEM and AFM images of bPrP(108-144), cPrP(108-144), huPrP(108-144)129M, and huPrP(108-144)129V fiber 31 4.3 CD spectroscopy 34 4.4 ThT titration assay 36 Chapter 5 Results Ⅲ. Cross-seeding assay of prion peptide fibers 39 5.1 Cross-seeding results of prion peptide fibers 39 Chapter 6 Results Ⅳ. Cryo-EM structure of bPrP(108-144) peptide fibrils 43 6.1 Data processing flow for structural analysis of bPrP(108-144) fibrils 43 6.1.1 Data collection statistics 43 6.1.2 Import 43 6.1.3 CTF Estimation 46 6.1.4 Manual picking 48 6.1.5 Particle extraction 49 6.1.6 2D classification 50 6.1.7 Particle extraction 52 6.1.8 Initial model building 53 6.1.9 3D refinement 55 6.1.10 3D classification 59 6.1.11 CTF refinement 61 6.1.12 3D refinement 63 6.1.13 3D classification 64 6.1.14 3D refinement, mask creation, and postprocessing 65 6.2 Fibril structure of bPrP(108-144) 68 6.2.1 Atomic modeling of bPrP(108-144) fibril 68 6.2.2 Structure details of bPrP(108-144) fibril 70 Chapter 7 Results Ⅴ. Cryo-EM structure of cPrP(108-144) peptide fibrils 74 7.1 Data processing flow for structural analysis of cPrP(108-144) fibrils 74 7.1.1 Data collection statistics 74 7.1.2 Import 75 7.1.3 CTF Estimation 75 7.1.4 Manual picking 77 7.1.5 Particle extraction 78 7.1.6 2D classification 79 7.1.7 Initial model building 79 7.1.8 3D refinement 82 7.1.9 3D classification 84 7.1.10 Type I fiber - 3D refinement 86 7.1.11 Type I fiber - CTF refinement 87 7.1.12 Type I fiber - 3D refinement, mask creation, and postprocessing 88 7.1.13 Type II fiber - 3D refinement 90 7.1.14 Type II fiber - CTF refinement 91 7.1.15 Type II fiber - 3D refinement, mask creation, and postprocessing 91 7.2 Fibril structure of cPrP(108-144) 95 7.2.1 Atomic modeling of cPrP(108-144) fibrils 95 7.2.2 Structure details of cPrP(108-144) fibrils 99 Chapter 8 Results Ⅵ. Structure comparation 107 Chapter 9 Discussion and future works 109 Reference 117 Appendix 123 | - |
| dc.language.iso | en | - |
| dc.subject | 物種傳播屏障 | zh_TW |
| dc.subject | 普利昂疾病 | zh_TW |
| dc.subject | 冷凍電子顯微鏡 | zh_TW |
| dc.subject | cryo-EM | en |
| dc.subject | prion disease | en |
| dc.subject | species barrier | en |
| dc.title | 普利昂胜肽序列差異對其纖維結構及交叉播種屏障的影響 | zh_TW |
| dc.title | Effect of Sequence Variation of Prion Peptides on Fibril Structure and Cross-seeding Barrier | en |
| dc.type | Thesis | - |
| dc.date.schoolyear | 113-2 | - |
| dc.description.degree | 碩士 | - |
| dc.contributor.oralexamcommittee | 陳振中;徐尚德 | zh_TW |
| dc.contributor.oralexamcommittee | Jerry Chun Chung Chan;Shang-Te Danny Hsu | en |
| dc.subject.keyword | 普利昂疾病,物種傳播屏障,冷凍電子顯微鏡, | zh_TW |
| dc.subject.keyword | prion disease,species barrier,cryo-EM, | en |
| dc.relation.page | 130 | - |
| dc.identifier.doi | 10.6342/NTU202502514 | - |
| dc.rights.note | 同意授權(限校園內公開) | - |
| dc.date.accepted | 2025-07-31 | - |
| dc.contributor.author-college | 生命科學院 | - |
| dc.contributor.author-dept | 生化科學研究所 | - |
| dc.date.embargo-lift | 2030-07-25 | - |
| 顯示於系所單位： | 生化科學研究所 | |
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