蛋白質與胜肽不正常堆疊在組織內形成斑塊是許多神經退性疾病的病徵之一，但由於這些錯誤堆疊蛋白質快速聚集的特性，難以觀測其堆疊過程與機制。在先前實驗室研究顯示，可能造成肌萎縮性脊髓側索硬化症(Amyotrophic lateral sclerosis, ALS)原因之一的蛋白質TDP-43，其C端胜肽片段D1 core具有快速聚集化形成類澱粉之特性。在這次研究中，我們目標建立一組化學的探針，此探針由雙硫鍵連結胜肽D1 core和細胞穿膜序列，當其進到細胞內，藉由細胞質內高還原電位的環境，產生自發性的氧化還原反應，使雙硫鍵斷裂，胜肽D1 core與細胞穿模序列分離後，其可展現自我聚集化的特性。我們利用固態液相胜肽合成法，結合出雙硫鍵胜肽探針JJS-1，於細胞外實驗中，使用還原劑穀胱甘肽模擬細胞質的環境，透過HPLC層析儀監測雙硫鍵裂解的過程；利用穿透式電子顯微鏡，觀測胜肽D1 core形成之類澱粉纖維狀聚集物。我們進一步將雙硫鍵的胜肽探針連接上螢光基團(Alexa Fluor 568 C5 Maleimide)，應用於細胞實驗中。從共軛焦螢光顯微鏡觀測下，雙硫鍵螢光胜肽探針JJS-2-AF568和JJS-3-AF568可以進入U2OS細胞中，並在細胞質形成帶有螢光的類澱粉狀之聚集體。於此，我們建立了能夠在細胞中釋放ALS疾病相關TDP-43蛋白片段的探針，並期待此探針未來能有更多的應用於觀察神經退化性疾病產生之機制。

Misfolding and accumulation of soluble protein into insoluble proteinaceous aggregates are the hallmark feature among many neurodegenerative diseases. Because misfolding peptide/proteins tend to aggregate rapidly, it is difficult to observe their stacking process and mechanism. Previously we have identified an amyloidogenic region (known as D1 core), from TDP-43 protein, a nuclear protein associated with amyotrophic lateral sclerosis (ALS). In this study, we aim to establish a disulfide-linked probe, which would allow the automatic release of the D1 core in the cytosol, where is mainly reducing environment, through native redox chemistry. To this end, we conjugate peptide D1 core to the cell-penetrating peptide with a disulfide bond in solid-phase peptide synthesis. In the presence of reductant glutathione, the cleavage of disulfide-linked probe JJS-1 into two fragments was confirmed by HPLC chromatography and mass spectrometry. In addition, the released D1 core moiety can spontaneously self-assemble into fibrils under transmission electron microscope (TEM). We also conjugated disulfide-linked probes to the fluorophore Alexa 568 C5 Maleimide for cellular study. Confocal microscopy imaging revealed that both the two probes JJS-2-AF568 and JJS-3-AF568 are cell permeable and rapidly develop into pronounced aggregates in the cytosol within U2OS cells. In current study, the probes which allows to automatically release the ALS-related TDP-43 peptides in cells have been created and we anticipate the further applications in deciphering the pathological mechanisms of neurodegenerative diseases.

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