研究TDP-43蛋白質中澱粉樣胜肽之聚集化特性;The Aggregation Properties of Amyloidogenic Peptides in TDP-43

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题名:	研究TDP-43蛋白質中澱粉樣胜肽之聚集化特性;The Aggregation Properties of Amyloidogenic Peptides in TDP-43
作者:	蔡侑儒;Tsai,Yu-Ju
贡献者:	化學學系
关键词:	聚集化;澱粉樣特性胜肽;TDP-43;Aggregation;Amyloidogenic Peptide;TDP-43
日期:	2016-07-07
上传时间:	2016-10-13 12:40:09 (UTC+8)
出版者:	國立中央大學
摘要:	蛋白質與胜肽因為不正常堆疊在組織內形成斑塊是許多神經退化性疾病的病徵。在2006年的時候，TAR DNA-binding Protein 43 (TDP-43) 被辨識出為肌萎縮性脊隨側索硬化症(Amyotrophic Lateral Sclerosis, ALS) 和額顳葉退化症 (Frontotemporal Lobar Degeneration, FTLD) 病理堆積物的主要成分。由於TDP-43蛋白質的C端與Sup35蛋白質的N端有一定的相似性，被推斷可能具有與Prion disease一樣強烈感染的特性。因此，為了驗證TDP-43是否具有類普恩蛋白 (Prion-like) 的特性，我們利用固相胜肽合成法 (Solid Phase Peptide Synthesis) 合成出富含麩醯胺酸 (Glutamine, Q) 和天門冬醯胺酸 (Asparagine, N) 的胜肽片段，然後對其做一系列澱粉樣蛋白特性以及引晶效應之測試，包括圓二色光譜、硫磺素T染色、穿透式電子顯微鏡觀測、無細胞表達系統以及細胞內效應，希望藉由觀測Q/N rich胜肽的特性使我們更為了解TDP-43蛋白質在ALS和FTLD所扮演的腳色。雖然在現今我們知道澱粉樣蛋白特性對於神經退化性疾病的病理學研究十分重要，然而對於澱粉樣蛋白特性在細胞或是組織內初期的特性卻尚未明朗，因此在本篇研究我們採取光控制的策略，設計了一條能夠利用紫外光驅動澱粉樣蛋白特性產生的胜肽，此胜肽在照光前能夠自組裝形成球體型態增加其進入細胞的能力。另外，藉由紫外光控制具有澱粉樣蛋白特性之胜肽在特定的時間及區域產生引晶效應，驅使細胞內的TDP-43蛋白質錯位 (mislocalized) 產生聚集化現象，這樣的設計使我們更加了解澱粉樣蛋白特性對於ALS和FTLD的影響。此種光控制胜肽的設計概念未來也能應用在其他神經退化性疾病，成為了一極具潛力的疾病模型。;The abnormal assembly of misfolded proteins or peptides into neurotoxic amyloid aggregates is a hallmark associated with many neurodegenerative diseases. In 2006, TAR DNA-binding protein (TDP-43) was first identified as a major component in the inclusions of patients with either amyotrophic lateral sclerosis (ALS) or frontotemporal lobar degeneration with ubiquitin-positive inclusion (FTLD-U). The strong tendency of the C-terminus of TDP-43 to form aggregates is likewise consistent with the behavior of a prion-related Q/N domain containing protein. In this study, we designed and synthesized the Q/N-rich polypeptides from the TDP-43 C-terminus with solid phase peptide synthesis (SPPS). Furthermore, their amyloid properties were characterized by electron microscopy (EM), circular dichroism (CD), thioflavin T assay, cell-free system and cultured cell observation. Although the importance of amyloid has been widely recognized in the pathogenesis of neurodegenerative diseases, the early phase of amyloidogenesis in cells or tissues remain obscure. To address this end, we employ a light-assisted strategy to develop a peptide that undergoes a fast amyloidogenesis upon UV illumination. Moreover, before photolysis, this photocontrollable peptide tends to self-assemble and adopt spherical vesicles, which is believed to confer cell-penetrating properties. Provided that the phototractable peptide can induce TDP-43 aggregates spatiotemporally, this design allows us to better understand amyloidogenesis in vivo. Such a light-assisted design has great potential to serve as a disease model of ALS or FTLD and seems applicable to other neurodegenerative diseases.
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