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研究生： 方裕勝
Fang, Yu-Sheng.
論文名稱： 蛋白質TDP-43寡聚體及其結構專一性抗體在生化和磷酸化的研究
The biochemical and phosphorylation studies of TDP-43 oligomers and its conformational antibody
指導教授： 陳韻如
Chen, Yun-Ru
蘇士哲
Sue, Shih-Che
口試委員: 袁小琀
Yuan, Hanna S.
黃聖言
Hwang, DennisW
黃介嶸
Huang, Jie-rong
學位類別： 博士
Doctor
系所名稱： 生命科學暨醫學院 - 生物資訊與結構生物研究所
Institute of Bioinformatics and Structural Biology
論文出版年： 2018
畢業學年度： 106
語文別： 英文
論文頁數： 87
中文關鍵詞： 神經退化性疾病
相關次數： 點閱：4  下載：0 
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    • 自從TDP-43被發現存在於腦前側額顳葉退化症(FTLD)和漸凍人症(ALS)的神經細胞沉澱物中，這個蛋白就開始被許多人研究。TDP-43由414個胺基酸所組成其中有兩個RNA recognition motifs (RRMs) 以及高度無序的 GQN-rich 在蛋白質的C端。大部分正常的TDP-43存在於細胞核中，負責基因調控的功能。到目前為止我們對TDP-43造成疾病的機制並不清楚，但病人的細胞中所找到的沉澱物，許多是ubiquitination，超磷酸化以及 N端缺失的蛋白質片段。目前研究能在爭論到底造成疾病的關鍵是因為TDP-43失去原有的功能史的細胞功能異常，抑或是不正常的TDP-43形成具有毒性的特殊物質使的細胞不正常？
    為了要更了解TDP-43，我們用重組蛋白的方式在大腸桿菌中產生並純化該蛋白。研究中我們發現TDP-43會形成一種特別的寡聚體，並且會被辨認類澱粉蛋白的寡聚體的抗體所辨認。此外，我們還發現TDP-43寡聚體會誘導乙型類澱粉蛋白形成寡聚體並抑制其纖維化的產生。另外，我們還製造了專門辨認TDP-43寡聚體的抗體，此抗體也辨認到在人類及老鼠的神經細胞中的沉澱物，我們還利用此抗體把組織中的TDP-43寡聚體純化出來並用電子顯微鏡觀察其形態。再者，TDP-43的磷酸化研究中我們發現C端的磷酸化會提高TDP-43的水溶性，但是其液態的相分離現象會降低。另外，我們還發現磷酸化的TDP-43會形成類澱粉纖維。
    總而言之，我們的研究發現TDP-43寡聚體的存在，並且磷酸化可能會導致類澱粉纖維的產生，提供了一個觀點去了解TDP-43跟類澱粉的致病機制有關，並期望之後的研究可以發展出診斷或是治療此疾病的方法。

    TAR DNA-binding protein-43, TDP-43, discovered in the two neurodegenerative diseases, frontal temporal lobar dementia (FTLD) and amyotrophic lateral sclerosis (ALS), is an important disease protein to be studied. TDP-43 contains 414 residues with two RNA recognition motifs (RRMs) and a highly disordered GQN-rich region in C-terminus. TDP-43 is mainly located in the nucleus and involved in gene regulation. The pathogenic mechanism of TDP-43 in the diseases is not clear. In the diseases TDP-43 inclusions were characterized with ubiquitination, hyper-phosphorylation and N-terminal truncation. Whether loss of normal function or gain of toxic function contributes to the pathogenic mechanism is still under debate.
    To study the mechanisms of TDP-43 in diseases, we generated recombinant human TDP-43 from E. coil. Size exclusion chromatography showed TDP-43 formed large-molecular-weight aggregates. Dynamic light scattering and transmission electron microscopy showed most of the particle populated at ~50 nm in diameter. Interestingly, TDP-43 was able to be recognized by specific anti-amyloid oligomer antibody. It is suggested that TDP-43 oligomers and amyloid oligomers have some similar epitopes. Besides, TDP43 oligomers induced synthetic Aβ40 to oligomer by inhibiting the amyloid fibril formation. We further generated polyclonal and monoclonal TDP-43 oligomer specific antibodies, TDP-O. Immunocytochemistry and immunohistochemistry showed polyclonal TDP-O antibody can stain inclusions in FTLD patient’s brain. Immunoprecipitation pulled down the TDP-43 oligomers from the extract of FTLD patient’s brain. In C-terminal phosphorylated TDP-43 study, we found C-terminal phosphorylation can increase its solubility. Furthermore, C-terminal serine phosphorylation of TDP-43 forms ThT positive amyloid fibril. In conclusion, our studies demonstrate that the full-length TDP-43 formed structurally stable amyloid-like oligomers and C-terminal phosphorylation leads to form amyloid fibril. Besides, our results supposed a possible mechanism for TDP-43 protienopathies which may be related to amyloidosis.

    Chapter 1 Introduction 1 1.1. Neurodegenerative diseases 1 1.1.1. Frontotemporal lobar degeneration 1 1.1.2. Amyotrophic lateral sclerosis 2 1.2. TDP-43 pathology 4 1.3. The structure of TDP-43 5 1.4. Amyloid and amyloidsis 6 1.5. TDP-43 is not a classic amyloid protein 7 1.6. Conformational specific antibody 8 1.7. Liquid-liquid phase separation 9 1.8. Hyper-phosphorylation in TDP-43 9 Chapter 2 Materials and Methods 11 2.1. Materials 11 2.2. Methods 13 2.2.1. Cloning 13 2.2.2. Protein expression 14 2.2.3. Protein purification (soluble) 14 2.2.4. Protein purification (insoluble) 15 2.2.5. Protein refolding 16 2.2.6. TDP-43 quantification 16 2.2.7. TDP-43 protein dialysis 18 2.2.8. Far-UV circular dichroism spectroscopy 18 2.2.9. Fluorescence spectroscopy 18 2.2.10. Dynamic Light Scatterling 19 2.2.11. Size exclusion chromatography 19 2.2.12. Denaturing assay 19 2.2.13. Aβ aggregation and cross-seeding assay 20 2.2.14. Transmission Electron Microscopy (TEM) 20 2.2.15. Immuno gold staining 20 2.2.16. Antibody production 21 2.2.17. Antibody purification 22 2.2.18. Indirect ELISA for TDP-43 22 2.2.19. Oligomers and monomers preparation 23 2.2.20. Competitive ELISA 23 2.2.21. Brain fractioning and immunoprecipitation 24 2.2.22. Liquid-liquid phase separation (LLPS) 25 2.2.23. TDP-43 aggregation 25 2.2.24. TDP-43 phosphorylation 26 2.2.25. Liposome leakage assay 26 Chapter 3 Characterization of TDP-43 oligomer 28 3.1. Soluble TDP-43 forms high-molecular-weight aggregates 28 3.2. TDP-43 oligomer can be recognized by anti-amyloid oligomer antibody 29 3.3. TDP-43 oligomer showed spherical shape 31 3.4. Secondary structure, hydrophobicity analysis and DNA bindind assay of TDP-43 oligomers 32 3.5. TDP-43 can induce Aβ40 oligomer formation by cross-seeding effect 36 Chapter 4 Anti-TDP-43 oligomer antibody 39 4.1. Generating anti-TDP-43 oligomer antibody, TDP-O 39 4.2. Characterization of polyclonal TDP-O antibody 39 4.3. TDP-43 oligomer in neurodegenerative disease 40 4.4. Monoclonal antibody TDP-O 47 4.5. Competitive ELISA 49 Chapter 5 C-terminal phosphorylation of TDP-43 51 5.1. Generating TDP-43 C-terminal phosphorylation mimicking mutants 51 5.2. Formation of WT, S5D, and S5A oligomers 52 5.3. Spectra characterization of TDP-43 variants 55 5.4. LLPS of TDP-43 variants 56 5.5. ThT assay of TDP-43 variants 59 5.6. Liposome leakage by TDP-43 variants 62 Chapter 6 Comparison of soluble and refolded TDP-43 64 6.1. Absorbance spectra 64 6.2. Circular dichroism 65 6.3. Bis-ANS spectra 66 Chapter 7 Disscussion 68 7.1. TDP-43 is not a classic amyloid 68 7.2. TDP-43 oligomer 69 7.3. The development of TDP-O antibody 70 7.4. TDP-43 liquid-liquid phase separation (LLPS) 70 7.5. C-terminal phosphorylation in TDP-43 71 7.6. The interaction with TDP-43 and lipid 73 7.7. Comparison of TDP-43 from refolded and soluble methods 74 References 75 Appendix 85

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