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標題: | 研究TDP-43變異胜肽片段於形成類澱粉樣纖維及細胞毒性的影響 The impact of mutations in TDP-43 fragments on the formation of amyloid fibers and cellular toxicity. |
作者: | Yu-Hsiang Wang 王鈺湘 |
指導教授: | 陳振中,黃人則 |
關鍵字: | TDP-43,類澱粉樣纖維,細胞毒性, TDP-43,amyloid fibers,cellular toxicity., |
出版年 : | 2012 |
學位: | 碩士 |
摘要: | 在2006年時,TAR DNA-binding protein 43 ( TDP-43 ) 被鑑定為肌萎縮性脊髓側索硬化症 ( amyotrophic lateral sclerosis;ALS ) 和額顳葉變性 ( frontotemporal lobar degeneration;FTLD ) 病理特徵堆積物的主要成分。其中,ALS是常見的神經退化性疾病之一,死亡率高居神經退化性疾病的第三名,僅次於阿茲海默症及帕金森氏症。然而,目前尚未有完全治癒其病徵的方法。 TDP-43位於神經的細胞核內,是由414個胺基酸及四個片段所構成,由N端往C端依次為N-terminus、RNA-recognition motif 1 ( RRM1)、RNA-recognition motif 2 ( RRM2 ) 及 C-terminus。近期研究指出,在ALS患者體內找到許多不同TDP-43蛋白質基因變異位置,可以發現多數變異集中於C端區域,證明了C端在TDP-43蛋白質變異上,具有相當的重要性。先前的研究結果顯示,將TDP-43的C端切成四個片段 ( D1-D4 ) ,令人感到有趣的是,實驗結果發現在磷酸鈉緩衝溶液 ( PBS ) 培養兩個星期之後,只有D1片段呈現出具纖維結構的聚集體,是屬於β類澱粉纖維沉積。為了更進一步探討TDP-43 的C端的變異位置在ALS的病人中所扮演的角色,我們成功的利用固相胜肽合成儀 ( SPPS ) 合成了各種不同的胜肽片段,包含了不具有病理突變的D1、具有病理突變的G294A ( sporadic )、G294V ( familiar)、G295S (sporadic ) 以及人工設計 (非病理) 的變異胜肽G294P、GGG294PPP以及GGG308PPP,探討其結構與不正常聚集體沉澱及纖維形成之間的關係,以及如何阻止不正常聚集體沉澱的發生。我們發現在穿透式電子顯微鏡 ( TEM ) 、圓二色光譜儀 ( CD )、 Thioflavin T ( ThT ) 染色實驗及拉曼光譜儀的觀察下,含有病理突變的胜肽 ( G294A、G294V、G295S ) 都會形成屬於β-sheet 結構的纏繞纖維構形聚集。並且,於沉降快慢分析法 ( time-course sedimentation assay ) 實驗結果,含有病理突變的胜肽片段具有細胞毒性且可調控加速纖維結構聚集化的現象。我們也發現在嵌入proline於D1序列中的胜肽 ( G294P、GGG294PPP ) ,會阻礙β-sheet結構形成以及減速類澱粉纖維的堆積。令人感到驚訝的是, GGG308PPP 胜肽片段,不僅阻止了類澱粉樣纖維結構聚集的產生,並且對Neuro2A細胞存活幾乎是沒有影響,不具毒性。我們推論特別是在D1 序列中308-310的核心 ( core ) 區域,嵌入proline可阻礙類澱粉狀纖維結構的形成。由此可推論此核心區域對於D1在形成類澱粉狀纖維結構上扮演很重要的角色。對於神經退化性疾病領域的研究,可以說是打開了一扇嶄新的大門。 TAR DNA-binding protein (TDP-43) has been identified as the major ubiqitinated component deposited in the inclusion bodies in Amyotrophic Lateral Sclerosis (ALS) and frontotemporal lobar degeneration (FTLD) since 2006. ALS is the third common neuron degenerative cause of adult death, after AD and PD. However, the exhaustive mechanism and the possible treatment for this disease remained unavailable. TDP-43 contained 414 residues. It has two RNA recognition motifs flanking by the N-terminal and C-terminus domains. Recent literatures have identified thirty dominant mutations in C-terminus glycine-rich domain of TDP-43 in sporadic and familial ALS patients. In order to understand the role of C-terminal domain of TDP-43 in ALS. Previously, we have shown that the peptide fragment D1 (region 287-322), derived from the TDP-43 C-terminus, may form beta-amyloid structures in the phosphate buffer. To characterize the impact of mutations on the amyloid formation, we have synthesized various peptides bearing pathogenic or de-novo designed mutations. We had discovered that peptides with pathological mutation (G294A, G294V, G295S) were all able to form twisted fibrils that belong to amyloid as confirmed by electron microscopy (EM), circular dichroism (CD), Thioflavin T (ThT) assay, and Raman spectroscopy. Moverover, pathological mutants had been shown to process their cellular toxicity to Neuro2A cell and speed up the aggregation process. We had also shown that introdution of proline in D1 (G294P and GGG294PPP), which is known to obstruct the β-sheet formation, may retard the amyloidogenesis and reduce their toxicity to neuronal cell. Surprisingly, GGG308PPP mutant could neither form beta-amyloid nor jeopardize cell survival, which hint on the importance of the glycines (308-310) during amyloidogenesis. Here, we have demonstrated the impact of pathological mutations in regulating fibril formation as well as cellular toxicity. The core sequence for amyloid formation is also characterized. |
URI: | http://tdr.lib.ntu.edu.tw/jspui/handle/123456789/16018 |
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