Tau蛋白寡聚體的製備與特性研究

Preparation and characterization of Tau protein oligomers

許詠翔(Yung-Hsiang Hsu)

指導教授 ： 陳振中

國立臺灣大學/理學院/化學系/碩士(2022年)

https://doi.org/10.6342/NTU202203914

摘要

阿茲海默症目前仍是一種無法治療的神經退化性疾病，由過度磷酸化之Tau蛋白所形成的神經纖維糾結是其病理特徵之一。近年來有越來越多證據指出，僅神經纖維糾結的形成就足以導致神經退化。纖維形成之前的中間態寡聚體具有更強的細胞毒性，且Tau寡聚體可能在神經元之間傳遞並誘導相鄰細胞中的Tau蛋白錯誤折疊，這對於阿茲海默症的形成具有關鍵性的病理意義，而本研究的最終目標是開發抑制寡聚體形成或促進清除寡聚體的藥物。目前對於Tau寡聚體的定義與大小仍不明確，以及寡聚體的不穩定性，使其難以研究。儘管過去研究中已經有不同方法來製備Tau寡聚體，但要製備與阿茲海默症患者構型相似的重組Tau寡聚體仍然是一個巨大的挑戰。現在用於探測Tau寡聚體構型的唯一可用工具是構型專一性抗體。在本研究中，我們從大腸桿菌表達系統製備出非常高純度的tau蛋白（2N4R，441 aa），並以肝素誘導Tau蛋白錯誤折疊，而肝素是一種已知能觸發Tau蛋白聚集的聚陰離子。我們優化了誘導條件，以最大化寡聚體的產率，透過粒徑篩析層析法（SEC）測定，並獲得了一段連續分佈的寡聚體，範圍從300 KDa到3 MDa（7-mer到70-mer），具有和阿茲海默症患者腦中寡聚體相似的構型抗原決定位，能被構型專一性Tau抗體Alz-50、TOMA和T22辨認。在粒徑篩析層析光譜圖中，Tau寡聚體波峰位置對應於斯托克斯半徑（Rs）18.0 nm，此Tau寡聚體在分析級超高速離心下，沉降曲線在24.5S左右達到峰值。且透過穿透式電子顯微鏡觀察到Tau寡聚體為顆粒狀聚集體，而不是棒狀原纖維。根據Siegel-Monte公式分析，我們製備佔比最多的寡聚體分子量為1.85 MDa（~40 mer），類似於從阿茲海默症患者腦中分離出的Tau寡聚體的大小。透過開發這些方法，我們以合理的產率獲得了毫克級約20-40個蛋白質分子組成的顆粒狀Tau寡聚體。它們具有類似於阿茲海默症患者大腦中發現的Tau寡聚體的構型抗原決定位，這有助於未來針對tau寡聚體的藥物開發。

關鍵字

Tau寡聚體 ； 肝素 ； 粒徑篩析層析法 ； 構形辨識抗體 ； 分析級超高速離心 ； 穿透式電子顯微鏡

並列摘要

Alzheimer's disease (AD) is still an untreatable neurodegenerative disease. Neurofibrillary tangles (NFTs) formed by hyperphosphorylated tau protein is one of its key pathological features. In recent years, increasing evidence has shown that the formation of neurofibrillary tangles alone is sufficient to cause neurodegeneration. The oligomeric intermediates that precede fibril formation have stronger cytotoxicity, and tau oligomers may transmit between neurons and induce tau misfolding in neighboring cells, a key pathological process in the progression of AD. The ultimate goal of this study is to develop drugs that can inhibit oligomer formation or promote oligomer clearance. At present, the definition of tau oligomers and their size range remain unclear. The instability of tau oligomers makes them difficult to study. Although different methods for preparing tau oligomers have been previously reported, it remains a great challenge to prepare recombinant tau oligomers with similar conformations as those found in AD patients. Currently, the only available tools to probe tau oligomer conformations are conformation-specific antibodies. In this study, we started with very high purity tau proteins (2N4R, 441 aa) prepared from E. coli expression system. Tau misfolding was induced by heparin, a polyanion known to trigger tau aggregation. We optimized the induction condition to maximize the yield of oligomeric species, as determined by size-exclusion chromatography (SEC). We obtained a continuum of oligomeric species that range from 300 KDa to 3 MDa (7-mer to 70-mer), and they share conformational epitopes found on oligomers from AD patient brains, recognized by conformational tau antibodies Alz-50, TOMA, and T22. The tau oligomer peak position in the SEC chromatogram corresponded to the Stokes radius (Rs) of 18.0 nm. When this peak fraction was submitted to analytical ultracentrifugation, the sedimentation profile peaked around 24.5S. Under transmission electron microscopy, they appeared as granular aggregates rather than rod-like protofibrils. According to Siegel-Monte analysis, the most abundant species in our preparation has a molecular weight of 1.85 MDa (~40-mer), similar to the size of tau oligomers isolated from AD patient brains. By developing these protocols, we obtained granular tau oligomers with around 20-40 protein units at milligram scale with a reasonable yield. They carry conformational epitopes similar to tau oligomers found in AD patient brains, which is useful for future drug development targeting tau oligomers.

並列關鍵字

Tau oligomer ； heparin ； size-exclusion chromatography ； conformational antibody ； analytical ultracentrifugation ； transmission electron microscopy

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