Proximity of SCG10 and prion protein in membrane rafts
Proximity of SCG10 and prion protein in membrane rafts
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DOI：
10.1111/jnc.13488
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发表时间：
2016-03-01
影响因子：
4.7
通讯作者：
Yokoyama, Takashi
中科院分区：
文献类型：
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作者：
Iwamaru, Yoshifumi;Kitani, Hiroshi;Yokoyama, Takashi
正常细胞朊蛋白（PrPC）转化为致病性朊蛋白（PrPSc）是朊病毒致病的重要过程。在培养模型中，膜筏被认为在PrPSc形成中起着关键作用。为了确定能够与PrPC相互作用并促进PrPSc在膜筏中形成的候选分子，我们采用了一种新的生化标记方法，称为酶介导的自由基源激活。将自由基源的酶介导的活化应用于来自小鼠神经母细胞瘤（N2 a）细胞的Lubrol WX不溶性洗涤剂抗性膜组分，其中表面PrPC用HRP缀合的抗PrP抗体标记。这些制剂的二维蛋白质印迹显示约20 kDa的生物素化斑点，等电点为8.0-9.0。液相色谱-串联质谱分析导致识别含有神经元特异性微管调节剂SCG 10的肽。SCG 10和PrPC的邻近使用邻近连接测定和免疫共沉淀测定来确认。用SCG 10小干扰RNA转染持续性22 L朊病毒感染的N2 a细胞降低了SCG 10的表达，但没有阻止PrPSc的积累，表明SCG 10似乎与PrPSc形成22 L朊病毒无关。免疫荧光和蛋白质印迹分析显示，在朊病毒感染的小鼠海马中SCG 10水平降低，表明SCG 10水平和朊病毒神经发病机制之间可能存在关联。
The conversion of normal cellular prion protein (PrPC) into its pathogenic isoform (PrPSc) is an essential event in prion pathogenesis. In culture models, membrane rafts are suggested to play a critical role in PrPSc formation. To identify the candidate molecules capable of interacting with PrPC and facilitating PrPSc formation in membrane rafts, we applied a novel biochemical labeling method termed enzyme-mediated activation of radical sources. Enzyme-mediated activation of radical sources was applied to the Lubrol WX insoluble detergent-resistant membrane fractions from mouse neuroblastoma (N2a) cells in which the surface PrPC was labeled with HRP-conjugated anti-PrP antibody. Two-dimensional western blots of these preparations revealed biotinylated spots of approximately 20 kDa with an isoelectric point of 8.0-9.0. Liquid chromatography-tandem mass spectrometry analysis resulted in the identification of peptides containing SCG10, the neuron-specific microtubule regulator. Proximity of SCG10 and PrPC was confirmed using proximity ligation assay and co-immunoprecipitation assay. Transfection of persistently 22L prion-infected N2a cells with SCG10 small interfering RNA reduced SCG10 expression, but did not prevent PrPSc accumulation, indicating that SCG10 appears to be unrelated to PrPSc formation of 22L prion. Immunofluorescence and western blot analyses showed reduced levels of SCG10 in the hippocampus of prion-infected mice, suggesting a possible association between SCG10 levels and the prion neuropathogenesis.