以胜肽探針研究漸凍人症中聚甘胺酸-丙胺酸雙肽蛋白質以及TDP-43蛋白質之病理現象

Induction of Glycine-alanine Dipeptide Repeats and TDP-43 Proteinopathies by Peptide Probes for ALS Studies

簡鴻名(Hung-Ming Chien)

指導教授 ： 陳玉如

共同指導教授 ： 黃人則(Jen-Tse Huang)

國立臺灣大學/理學院/化學研究所/博士(2021年)

https://doi.org/10.6342/NTU202102115

摘要

在腦中所發現之蛋白質包含體為漸凍人症候群中的主要病徵，其亦被懷疑與疾病的發展有關。而TDP-43蛋白質作為病患組織中蛋白質包含體的主要成分，其經常被發現具有過度磷酸化、過度泛素化之現象，同時此蛋白質亦可進一步遭受裁剪而成具類澱粉蛋白性質之片段。除了TDP-43蛋白質以外，近期的研究發現自第九號染色體之第七十二號開放閱讀框突變基因轉譯之甘胺酸-丙胺酸雙肽蛋白質(Glycine-alanine dipeptide repeats)亦沉積於病患之神經元中，且其亦具類澱粉蛋白質之特性。隨著越來越多的研究表明TDP-43蛋白質與甘胺酸-丙胺酸雙肽蛋白質於疾病中可能扮演著重要的角色，針對這些蛋白質的定性實驗亦顯得更加重要。然而，這些蛋白質的生物物理與生物化學性質之研究卻由於其溶解度與快速聚集等因素而備受阻礙。
為了克服這些困難以研究這些蛋白質之特性，我們設計並利用固相化學方法合成了一系列的胜肽探針，其由甘胺酸-丙胺酸雙肽蛋白質片段、八離胺酸肽片段(Octalysine)、與甲氧基硝基苯(Methoxynitrobenzene)衍生物作為橋接所組成。這些探針可有效的增進甘胺酸-丙胺酸雙肽蛋白質片段之溶解度、預防其片段自我聚集、並能夠於細胞中藉由紫外光照實行可控制之釋放。利用此探針並結合顯微鏡技術，我們發現甘胺酸-丙胺酸雙肽蛋白質片段會先行組裝成類澱粉蛋白質寡聚物(Amyloid oligomer)並進一步聚集成類澱粉蛋白質纖維；此胜肽探針亦可幫助釐清甘胺酸-丙胺酸雙肽蛋白質片段之詳細生物物理與生物化學特性。另外，藉由表達可於核質穿梭之螢光蛋白質於細胞系統內，我們發現到甘胺酸-丙胺酸雙肽蛋白質片段會導致細胞內之核質運輸功能異常。而利用電子顯微鏡觀察探針處理過後之細胞切片，我們亦注意到此片段會引起細胞核膜之內陷與不正常之折疊；另一方面，於進一步的免疫染色與生物化學實驗中，我們亦證實了甘胺酸-丙胺酸雙肽蛋白質片段類澱粉蛋白質寡聚物可引起脂質膜之穿透性增加。我們亦於後續的細胞實驗中發現甘胺酸-丙胺酸雙肽蛋白質片段可引起TDP-43蛋白質於細胞質內之不正常堆積，其現象與先前文獻所報導之病理特徵相似。
事實上，除了甘胺酸-丙胺酸雙肽蛋白質外，亦有研究證明了TDP-43蛋白質羧基端之裁剪片段亦可於細胞內引起TDP-43之蛋白質病理。因此，我們設計並合成了於還原環境中藉由雙硫鍵觸發之自釋放胜肽探針以研究TDP-43蛋白質羧基端之裁剪片段之堆疊。我們於此研究中仔細地觀測其胜肽探針裂解過程與釋放出之TDP-43片段之類澱粉蛋白質特性。
總結來說，我們成功利用甲氧基硝基苯或雙硫鍵之化學以設計並製備了一系列之胜肽探針。我們更進一步於其相對應之系統中，詳細觀測了TDP-43蛋白質裁剪片段與甘胺酸-丙胺酸雙肽蛋白質片段之生物物理與生物化學性質。我們期許在未來可藉由這些蛋白質片段之定性研究成果來幫助釐清漸凍人疾病發生之病因。

關鍵字

漸凍人症 ； C9orf72 ； 類澱粉蛋白質 ； 胜肽探針 ； 甘胺酸-丙胺酸雙肽蛋白質 ； TDP-43蛋白質

並列摘要

Protein inclusions in the brains with amyotrophic lateral sclerosis (ALS) have been suggested as the most common pathological hallmark and correlated with disease progression. As a major component of pathological inclusions, transactive response DNA binding protein 43 (TDP-43) was hyperphosphorylated, hyperubiquitinated, and further truncated into fragments with amyloid properties in patient. In addition to TDP-43, glycine-alanine dipeptide repeats (GA DPRs) translated from the mutated chromosome 9 open reading frame 72 (C9orf72) gene has been recently identified in the cytoplasm of neurons with amyloid properties. Though collective evidence has demonstrated that both TDP-43 and GA DPRs inclusions may play pivotal roles in the disease pathology, their detailed biophysical and biochemical characterization were impeded due to poor solubility and rapid polymerization nature.
To overcome these difficulties, we have synthesized a series of chemical probes composed of a GA DPRs fragment and a octalysine sequence linking by a methoxynitrobenzene photolinker. These probes could efficiently enhance the solubility of GA DPRs, prevent them from self-assembly, and enable the controllable liberation in cells upon irradiation of UV light. By employing these probes and advanced microscopies, we have shown GA DPRs first assemble into amyloid oligomers and then evolve into amyloid fibrils. The detailed biophysical and amyloid properties of GA DPRs were characterized. By the ectopic expression of shuttling reporter protein in neuron-like cells, we demonstrated GA DPRs cause the nucleocytoplasmic transport dysregulation. In this regard, we also noticed GADPRs have caused the nuclear depletion of Ran protein and cytosolic diffusion of importin-β. Additionally, GA DPRs released from the probe were found to cause the invagination of nuclear envelope by transmission electron microscopy observation. We further identified it is the GA DPRs amyloid oligomers rather than fibrils permeabilize the lipid membrane through immunohistochemistry and biochemical assays. Moreover, we noticed GA DPRs could lead to TDP-43 cytosolic retention in mouse cortical neurons, which is consistent with the previous studies.
In fact, it has also been shown that TDP-43 C-terminal peptide fragments could initiate TDP-43 proteinopathy in addition to GA DPRs. Therefore, we also prepared a disulfide bond triggering auto-releasing probe designed for the liberation of TDP-43 fragment upon reducing environment. The detailed cleavage process and the amyloidogenesis properties were monitored in this study and the following cellular studies are still ongoing now.
Conclusively, we have successfully designed and synthesized peptide probes on the basis of either methoxynitrobenzene photochemistry or disulfide bond chemistry. By applying these probes on suitable platforms, we explored the biophysical properties and detailed proteinopathy of GA DPRs and TDP-43 C-terminal fragment, which may eventually shed light on ALS pathology in the future.

並列關鍵字

Amyotrophic lateral sclerosis ； C9orf72 ； Amyloid ； Peptide probe ； Glycine-alanine dipeptide repeats ； TDP-43

參考文獻

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