利用籠化TDP-43胜肽片段觀察光引導類澱粉纖維形成之過程

Monitoring the light-induced amyliodogenesis with caged TDP-43 peptides

趙書漢(Shu-Han Chao)

指導教授 ： 李賢明 ； 謝發坤

國立中央大學/理學院/化學系/碩士(2015年)

摘要

目前許多文獻以光控物件作為基礎，建立各種控制釋放研究已經顯而易見。實驗室先前研究成果，顯示TDP-43蛋白質的C端胜肽片段D1之核心D1core具有快速聚集化形成類澱粉纖維之特性。本研究透過光控類澱粉纖維形成之概念與細胞穿膜序列作結合將D1core與多個精胺酸（arginine, R）以非天然光裂解胺基酸作連結，形成一段可光控形成類澱粉纖維之胜肽（8R-PL-D1core），此非天然光裂解胺基酸是以硝基苯為主體架構的光化學分子，透過照射紫外光使硝基苯產生自由基反應，進而引導胺基斷裂。將胜肽培養一段時間後，使用圓二色光譜儀鑑別其照光前後之二級結構；穿透式電子顯微鏡觀察其照光前後構形的變化；可見光紫外光光譜儀偵測其照光前後混濁度之差異，顯示多精胺酸能有效抑制D1core的聚集化速度，因此在光解前後此胜肽聚集化形成類澱粉纖維之速度會有極大差異，在結構上的轉變也會相差許多。進一步於活細胞實驗中，先將8R-PL-D1core合成上螢光基團（Alexa flour 568®）後，再加入細胞中進行觀察。擁有多精胺酸做為穿細胞膜序列，此可光控形成類澱粉纖維之胜肽可以順利進入細胞內，並與在細胞質中的TDP-43蛋白質相互作用。透過足夠UV光的照射，D1core失去多精胺酸抑制其聚集化形成類澱粉纖維，將與細胞質內的TDP-43蛋白質互相作用，並經由引晶效應（seeding effect）使TDP-43蛋白質聚集化而形成聚集體。此可光控形成類澱粉纖維之胜肽加入神經細胞實驗後極有潛力能成為一個ALS在疾病上的模型（disease model），且能夠期待它應用於觀察神經退化性疾病產生之機制。

關鍵字

胜肽

並列摘要

Photolytic device has been used widely in the controlled release of chemicals. Previously we have identified the core sequence of D1 region of TDP-43’s C terminus (D1core) and characterized its rapid aggregation and amyloidogenesis. In this study, by adding a photolabile unnatural amino acid and eight mer positively charged arginine (8R) N terminally, we synthesized a photochemically inducible D1core peptide (8R-PL-D1core). Provided that 8R readily inhibits D1core aggregation, the amyloidogenicity of the peptide would be only activated upon UV-mediated photocleavage. From 1) the ultrastructural changes observed under a transmission electron microscope (TEM), 2) the secondary structure alteration revealed by circular dichroism (CD), and 3) the turbidity assessed by UV–visible spectrophotometer, the significant changes of the peptide after UV illumination were reported. In addition to the in vitro characterizations, the fluorophore Alexa 568 was conjugated to 8R-PL-D1core to learn its dynamics in neuronal cells since the 8R moiety also confers cell membrane permeability. Of note, only UV-mediated release of D1core is capable of triggering cytosolic TDP-43 to form amyloid fiber. Taken these together, this photochemically inducible peptide could serve as a simple but powerful disease model of ALS and its further application on neurodegenerative diseases is highly expected as well.

並列關鍵字

TDP-43

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