複雑怪奇なリン酸化タンパク質タウのPhos-tag解析  [in Japanese] Phos-tag SDS-PAGE analysis of complicated phosphorylation of a neurodegenerative disease protein tau  [in Japanese]

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Author(s)

    • 木村 妙子 Kimura Taeko
    • 放射線医学総合研究所・脳機能イメージング研究部|首都大学東京・理工学研究科・生命科学専攻 Department of Functional Brain Imaging Research, National Institute for Quantum and Radiological Science and Technology|Laboratory of Molecular Neuroscience, Department of Biological Sciences, Tokyo Metropolitan University
    • 久永 眞市 Hisanaga Shinichi
    • 首都大学東京・理工学研究科・生命科学専攻 Laboratory of Molecular Neuroscience, Department of Biological Sciences, Tokyo Metropolitan University

Abstract

<p>タウは脳で発現する微小管結合タンパク質であり,また,タウオパチーと呼ばれる神経変性疾患群では高リン酸化(40箇所以上)され,凝集体を形成している.タウのリン酸化については非常に多くの報告があるが,リン酸化の実態は明確ではない.というのは大部分がリン酸化抗体を用いた研究で,定量的リン酸化アイソタイプという概念が欠けている.そのため理解に苦しむような結果も多い.本研究内ではPhos-tag法を用いて多数のリン酸化アイソタイプとして存在する培養細胞タウから病態タウについての解析も行った.培養細胞に強制発現させたタウは10以上のアイソタイプとして存在し,前頭側頭葉型認知症(FTDP-17)のR406W変異タウは特定のリン酸化に影響を与えたこと,脳内では非リン酸化のタウがある程度存在することがわかった.Phos-tag法はリン酸化部位の多いタンパク質の解析にも有用であった.</p>

<p>Tau is a microtubule-associated protein primarily expressed in the axons of neurons. Tau regulates microtubule dynamics and transport of organelles along microtubules in axons. These functions of tau are regulated by phosphorylation with a number of protein kinases. Tau is also a major component of neurofibrillary tangles (NFTs) in the brains of patients with tauopathies, including Alzheimer's disease (AD), frontotemporal dementia and parkinsonism linked to chromosome 17 (FTDP-17). Tau in NFTs is hyperphosphorylated, and therefore, the phosphorylation of tau has been intensively investigated. To date, more than 40 phosphorylation sites have been identified in aggregated tau. However, the physiological and pathological roles of the respective phosphorylation sites and how their phosphorylation is regulated are unclear. This could be due to the lack of the proper method of analyzing tau phosphorylation. Most of previous phosphorylation studies were conducted with a repertory of phosphorylation-site specific antibodies. While they provide relative changes of respective phosphorylation sites, it is hard to estimate total or absolute phosphorylation. To overcome these problems, we have recently applied the Phos-tag technique to the analysis of tau phosphorylation. This method separated tau into many bands on SDS-PAGE depending on phosphorylation, making it possible to analyze tau phosphorylation quantitatively and combinatorially. We identified phosphor-isotypes of tau expressed in cultured cells and found the abundance of nonphosphorylated tau in mouse and human tauopathy brains. Our results demonstrate that the Phos-tag would be a powerful method to characterize in vivo physiological and pathological phosphorylation of tau.</p>

Journal

  • Electrophoresis Letters

    Electrophoresis Letters 61(2), 49-52, 2017

    Japanese Electrophoresis Society

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