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研究生: 許菀庭
Wan-Ting Hsu
論文名稱: 利用FUS蛋白低複雜度區域片段誘發液滴及類澱粉樣纖維之生成
Induction of Liquid Droplet and Amyloid-like Nanofibrils by the FUS Low Complexity Domain Fragment
指導教授: 黃人則
Jen-Tse Huang
何明樺
Ming-Hua Ho
口試委員: 黃人則
Jen-Tse Huang
何明樺
Ming-Hua Ho
陳振中
Chun-Chung Chan
學位類別: 碩士
Master
系所名稱: 工程學院 - 化學工程系
Department of Chemical Engineering
論文出版年: 2021
畢業學年度: 109
語文別: 中文
論文頁數: 73
中文關鍵詞: 液滴奈米纖維FUS蛋白低複雜度區域
外文關鍵詞: liquid droplet, nanofibrils, FUS protein, low complexity domain
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摘要 III ABSTRACT IV 致謝 V 目錄 VII 圖目錄 X 表目錄 XIII 附圖目錄 XIV 第1章 緒論 1 1.1 仿生胜肽經自我聚集成生物超分子 1 1.1.1 胜肽聚集成類澱粉奈米纖維 2 1.1.2 生物體中的液-液相分離 (Liquid Liquid Phase Separation, LLPS) 3 1.1.2.1 探討LLPS中的作用力 4 1.1.2.2 不同作用力模式下的刺激與回應 6 1.2 FUS (Fused in Sarcoma) 蛋白 8 1.3 研究動機與目的流程 11 第2章 實驗儀器與材料 13 2.1 實驗儀器 13 2.2 實驗材料 14 2.2.1 胜肽合成與純化試劑配置 14 2.2.2 藥品表 15 第3章 實驗方法 16 3.1 胜肽樣品製備 (合成、純化與鑑定) 16 3.1.1 固相胜肽合成法 (Solid-Phase Peptide Synthesis, SPPS) 16 3.1.2 高效液相層析 (High-Performance Liquid Chromatography, HPLC) 19 3.1.3 基質補助雷射脫附游離質譜法 (MALDI Mass Spectrometry) 20 3.2 胜肽材料 (material) 性質分析 21 3.2.1 混濁度測試 (Turbidity assay) 21 3.2.2 穿透式電子顯微鏡 (Transmission Electron Microscopy, TEM) 22 3.2.2.1 胜肽纖維負染步驟 23 3.2.3 半衰減全反射式傅立葉轉換紅外線光譜分析法 (Attenuated total reflectance Fourier-transform infrared spectroscopy, ATR-FTIR) 24 3.2.4 Thioflavin T染色螢光光譜 (ThT Fluorescence spectrometry) 25 3.2.5 微分干涉相差顯微技術 (Differential interference contrast microscopy, DIC) 26 3.2.6 全反射螢光影觀測 (Total internal reflection fluorescence, TIRF) 26 第4章 實驗結果 27 4.1 光控相轉換胜肽材料的設計 27 4.1.1 FUS蛋白纖維化片段之序列挑選 28 4.1.2 多正電荷序列及光控連接子的設計 30 4.2 光控制胜肽的合成、純化與鑑定 31 4.3 胜肽液-液相分離之性質鑑定 31 4.3.1 胜肽濃度與液滴大小、數量的關係 32 4.3.2 低溫有助於液滴生成 34 4.3.3 聚乙二醇有助於液滴生成 36 4.3.4 鹽離子抑制液滴形成 37 4.3.5 以穿透式電子顯微鏡觀察液滴之型態 38 4.4 以HPLC和MALDI - TOF監控光控胜肽之裂解反應 39 4.5 胜肽光解形成類澱粉樣纖維之型態分析 40 4.5.1 有無光解反應之光控胜肽JSF於穿透式電子顯微鏡形態 40 4.5.2 以衰減式全反射分析紅外線光譜 (ATR-FTIR) 分析胜肽纖維之二級結構 41 4.5.3 以全反射螢光影像觀測 (TIRF) 結合ThT染色證實為類澱粉樣纖維 43 4.6 以光控胜肽突變序列探討其液-液相分離能力 44 4.6.1 減少苯環數目抑制液滴形成 44 4.6.2 減少正電荷數目抑制液滴形成 45 4.6.3 以Lys取代Arg作為正電荷來源抑制液滴形成 46 第5章 實驗討論 47 第6章 總結 49 附圖 50 參考資料 56

1. Maji, S. K.; Schubert, D.; Rivier, C.; Lee, S.; Rivier, J. E.; Riek, R., Amyloid as a depot for the formulation of long-acting drugs. PLoS Biology 2008, 6 (2), e17.
2. Mankar, S.; Anoop, A.; Sen, S.; Maji, S. K., Nanomaterials: amyloids reflect their brighter side. Nano reviews 2011, 2 (1), 6032.
3. Chiti, F.; Dobson, C. M., Protein misfolding, functional amyloid, and human disease. Annual Review of Biochemistry 2006, 75, 333-366.
4. Quan, L.; Gu, J.; Lin, W.; Wei, Y.; Lin, Y.; Liu, L.; Ding, H.; Pan, C.; Xie, Z.; Wu, T., A BODIPY biosensor to detect and drive self-assembly of diphenylalanine. Chemical Communications 2019, 55 (59), 8564-8566.
5. Fan, T.; Yu, X.; Shen, B.; Sun, L., Peptide self-assembled nanostructures for drug delivery applications. Journal of Nanomaterials 2017, 2017.
6. Buchan, J. R.; Parker, R., Eukaryotic stress granules: the ins and outs of translation. Molecular cell 2009, 36 (6), 932-941.
7. Yoshizawa, T.; Nozawa, R.-S.; Jia, T. Z.; Saio, T.; Mori, E., Biological phase separation: cell biology meets biophysics. Biophysical reviews 2020, 12 (2), 519-539.
8. Markmiller, S.; Soltanieh, S.; Server, K. L.; Mak, R.; Jin, W.; Fang, M. Y.; Luo, E.-C.; Krach, F.; Yang, D.; Sen, A., Context-dependent and disease-specific diversity in protein interactions within stress granules. Cell 2018, 172 (3), 590-604. e13.
9. Darling, A. L.; Liu, Y.; Oldfield, C. J.; Uversky, V. N., Intrinsically disordered proteome of human membrane‐less organelles. Proteomics 2018, 18 (5-6), 1700193.
10. Uversky, V. N.; Gillespie, J. R.; Fink, A. L., Why are “natively unfolded” proteins unstructured under physiologic conditions? Proteins: structure, function, and bioinformatics 2000, 41 (3), 415-427.
11. Dignon, G. L.; Best, R. B.; Mittal, J., Biomolecular phase separation: From molecular driving forces to macroscopic properties. Annual review of physical chemistry 2020, 71, 53-75.
12. Levin, A.; Hakala, T. A.; Schnaider, L.; Bernardes, G. J.; Gazit, E.; Knowles, T. P., Biomimetic peptide self-assembly for functional materials. Nature Reviews Chemistry 2020, 4 (11), 615-634.
13. Aumiller, W. M.; Keating, C. D., Phosphorylation-mediated RNA/peptide complex coacervation as a model for intracellular liquid organelles. Nature chemistry 2016, 8 (2), 129-137.
14. Pak, C. W.; Kosno, M.; Holehouse, A. S.; Padrick, S. B.; Mittal, A.; Ali, R.; Yunus, A. A.; Liu, D. R.; Pappu, R. V.; Rosen, M. K., Sequence determinants of intracellular phase separation by complex coacervation of a disordered protein. Molecular cell 2016, 63 (1), 72-85.
15. Dougherty, D. A., Cation-π interactions involving aromatic amino acids. The Journal of nutrition 2007, 137 (6), 1504S-1508S.
16. Ma, J. C.; Dougherty, D. A., The cation− π interaction. Chemical reviews 1997, 97 (5), 1303-1324.
17. Hughes, M. P.; Sawaya, M. R.; Boyer, D. R.; Goldschmidt, L.; Rodriguez, J. A.; Cascio, D.; Chong, L.; Gonen, T.; Eisenberg, D. S., Atomic structures of low-complexity protein segments reveal kinked β sheets that assemble networks. Science 2018, 359 (6376), 698-701.
18. Gomes, E.; Shorter, J., The molecular language of membraneless organelles. Journal of Biological Chemistry 2019, 294 (18), 7115-7127.
19. Quiroz, F. G.; Chilkoti, A., Sequence heuristics to encode phase behaviour in intrinsically disordered protein polymers. Nature materials 2015, 14 (11), 1164-1171.
20. Niskanen, J.; Tenhu, H., How to manipulate the upper critical solution temperature (UCST)? Polymer Chemistry 2017, 8 (1), 220-232.
21. Kroschwald, S.; Munder, M. C.; Maharana, S.; Franzmann, T. M.; Richter, D.; Ruer, M.; Hyman, A. A.; Alberti, S., Different material states of Pub1 condensates define distinct modes of stress adaptation and recovery. Cell reports 2018, 23 (11), 3327-3339.
22. Vance, C.; Rogelj, B.; Hortobágyi, T.; De Vos, K. J.; Nishimura, A. L.; Sreedharan, J.; Hu, X.; Smith, B.; Ruddy, D.; Wright, P., Mutations in FUS, an RNA processing protein, cause familial amyotrophic lateral sclerosis type 6. Science 2009, 323 (5918), 1208-1211.
23. Bentmann, E.; Neumann, M.; Tahirovic, S.; Rodde, R.; Dormann, D.; Haass, C., Requirements for stress granule recruitment of fused in sarcoma (FUS) and TAR DNA-binding protein of 43 kDa (TDP-43). Journal of Biological Chemistry 2012, 287 (27), 23079-23094.
24. Bosco, D. A.; Lemay, N.; Ko, H. K.; Zhou, H.; Burke, C.; Kwiatkowski Jr, T. J.; Sapp, P.; McKenna-Yasek, D.; Brown Jr, R. H.; Hayward, L. J., Mutant FUS proteins that cause amyotrophic lateral sclerosis incorporate into stress granules. Human molecular genetics 2010, 19 (21), 4160-4175.
25. Patel, A.; Lee, H. O.; Jawerth, L.; Maharana, S.; Jahnel, M.; Hein, M. Y.; Stoynov, S.; Mahamid, J.; Saha, S.; Franzmann, T. M., A liquid-to-solid phase transition of the ALS protein FUS accelerated by disease mutation. Cell 2015, 162 (5), 1066-1077.
26. Guccione, E.; Richard, S., The regulation, functions and clinical relevance of arginine methylation. Nature reviews Molecular cell biology 2019, 20 (10), 642-657.
27. Monahan, Z.; Ryan, V. H.; Janke, A. M.; Burke, K. A.; Rhoads, S. N.; Zerze, G. H.; O'Meally, R.; Dignon, G. L.; Conicella, A. E.; Zheng, W., Phosphorylation of the FUS low‐complexity domain disrupts phase separation, aggregation, and toxicity. The EMBO journal 2017, 36 (20), 2951-2967.
28. Molliex, A.; Temirov, J.; Lee, J.; Coughlin, M.; Kanagaraj, A. P.; Kim, H. J.; Mittag, T.; Taylor, J. P., Phase separation by low complexity domains promotes stress granule assembly and drives pathological fibrillization. Cell 2015, 163 (1), 123-133.
29. Murray, D. T.; Kato, M.; Lin, Y.; Thurber, K. R.; Hung, I.; McKnight, S. L.; Tycko, R., Structure of FUS protein fibrils and its relevance to self-assembly and phase separation of low-complexity domains. Cell 2017, 171 (3), 615-627. e16.
30. Wang, J.; Choi, J.-M.; Holehouse, A. S.; Lee, H. O.; Zhang, X.; Jahnel, M.; Maharana, S.; Lemaitre, R.; Pozniakovsky, A.; Drechsel, D., A molecular grammar governing the driving forces for phase separation of prion-like RNA binding proteins. Cell 2018, 174 (3), 688-699. e16.
31. Qamar, S.; Wang, G.; Randle, S. J.; Ruggeri, F. S.; Varela, J. A.; Lin, J. Q.; Phillips, E. C.; Miyashita, A.; Williams, D.; Ströhl, F., FUS phase separation is modulated by a molecular chaperone and methylation of arginine cation-π interactions. Cell 2018, 173 (3), 720-734. e15.
32. Pedersen, S. L.; Tofteng, A. P.; Malik, L.; Jensen, K. J., Microwave heating in solid-phase peptide synthesis. Chemical Society Reviews 2012, 41 (5), 1826-1844.
33. Smolira, A.; Wessely-Szponder, J., Importance of the Matrix and the Matrix/Sample Ratio in MALDI-TOF-MS Analysis of Cathelicidins Obtained from Porcine Neutrophils. Appl. Biochem. Biotechnol. 2015, 175 (4), 2050-2065.
34. Biancalana, M.; Koide, S., Molecular mechanism of Thioflavin-T binding to amyloid fibrils. Biochimica et Biophysica Acta (BBA)-Proteins and Proteomics 2010, 1804 (7), 1405-1412.
35. Mutter, M.; Chandravarkar, A.; Boyat, C.; Lopez, J.; Dos Santos, S.; Mandal, B.; Mimna, R.; Murat, K.; Patiny, L.; Saucède, L., Switch peptides in statu nascendi: induction of conformational transitions relevant to degenerative diseases. Angewandte Chemie International Edition 2004, 43 (32), 4172-4178.
36. Nepomniaschiy, N.; Grimminger, V.; Cohen, A.; DiGiovanni, S.; Lashuel, H. A.; Brik, A., Switch peptide via Staudinger reaction. Organic letters 2008, 10 (22), 5243-5246.
37. He, R. Y.; Chao, S. H.; Tsai, Y. J.; Lee, C. C.; Yu, C. Y.; Gao, H. D.; Huang, Y. A.; Hwang, E.; Lee, H. M.; Huang, J. J., Photocontrollable Probe Spatiotemporally Induces Neurotoxic Fibrillar Aggregates and Impairs Nucleocytoplasmic Trafficking. ACS Nano 2017, 11 (7), 6795-6807.
38. Bochet, C. G., Photolabile protecting groups and linkers. Journal of the Chemical Society, Perkin Transactions 1 2002, (2), 125-142.
39. Paraskevopoulou, V.; Falcone, F. H., Polyionic tags as enhancers of protein solubility in recombinant protein expression. Microorganisms 2018, 6 (2), 47.
40. Minton, A. P., The influence of macromolecular crowding and macromolecular confinement on biochemical reactions in physiological media. Journal of biological chemistry 2001, 276 (14), 10577-10580.
41. Park, S.; Barnes, R.; Lin, Y.; Jeon, B.-j.; Najafi, S.; Delaney, K. T.; Fredrickson, G. H.; Shea, J.-E.; Hwang, D. S.; Han, S., Dehydration entropy drives liquid-liquid phase separation by molecular crowding. Communications Chemistry 2020, 3 (1), 1-12.
42. Priftis, D.; Tirrell, M., Phase behaviour and complex coacervation of aqueous polypeptide solutions. Soft Matter 2012, 8 (36), 9396-9405.
43. Kotormán, M.; Maria Simon, L.; Borics, A.; Szabo, M. R.; Szabó, K.; Szogi, T.; Fulop, L., Amyloid-like fibril formation by trypsin in aqueous ethanol. Inhibition of fibrillation by PEG. Protein and peptide letters 2015, 22 (12), 1104-1110.
44. Sadat, A.; Joye, I. J., Peak Fitting Applied to Fourier Transform Infrared and Raman Spectroscopic Analysis of Proteins. Applied Sciences 2020, 10 (17), 5918.
45. Waeytens, J.; Mathurin, J.; Deniset-Besseau, A.; Arluison, V.; Bousset, L.; Rezaei, H.; Raussens, V.; Dazzi, A., Probing amyloid fibril secondary structures by infrared nanospectroscopy: experimental and theoretical considerations. Analyst 2021, 146 (1), 132-145.
46. Gallivan, J. P.; Dougherty, D. A., Cation-π interactions in structural biology. Proceedings of the National Academy of Sciences 1999, 96 (17), 9459-9464.
47. Krainer, G.; Welsh, T. J.; Joseph, J. A.; Espinosa, J. R.; Wittmann, S.; de Csilléry, E.; Sridhar, A.; Toprakcioglu, Z.; Gudiškytė, G.; Czekalska, M. A., Reentrant liquid condensate phase of proteins is stabilized by hydrophobic and non-ionic interactions. Nature communications 2021, 12 (1), 1-14.

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