Nuclear import factor transportin and arginine methyltransferase 1 modify FUS neurotoxicity in Drosophila
Nuclear import factor transportin and arginine methyltransferase 1 modify FUS neurotoxicity in Drosophila
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DOI：
10.1016/j.nbd.2014.11.003
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发表时间：
2015-02-01
影响因子：
6.1
通讯作者：
Kahle, Philipp J.
中科院分区：
文献类型：
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作者：
Jaeckel, Sandra;Summerer, Anna K.;Kahle, Philipp J.
包含融合肉瘤（PUS）的包涵体见于家族性和散发性不可治愈的进行性运动神经元疾病肌萎缩侧索硬化症病例以及常见的痴呆形式额颞叶痴呆。大多数疾病相关突变位于PUS的C-末端脯氨酸-酪氨酸核定位序列（PY-NLS），并损害其核输入。在细胞培养中已经显示，PUS的核输入由转运蛋白介导，转运蛋白结合PY-NLS和FUS的最后一个精氨酸/甘氨酸/富含甘氨酸（RGG）结构域。蛋白质精氨酸甲基转移酶（PRMT）对最后一个RGG结构域的甲基化减弱了转运蛋白结合，因此损害了FUS的核转位。为了研究PUS在体内模型中的核输入的要求，我们产生了不同的转基因果蝇系，表达人FUS野生型（hFUS wt）和两种疾病相关变体P525 L和R495 X，其中NLS分别突变或完全缺失。为了排除由异源hFUS表达引起的影响，我们分析了果蝇FUS直系同源物Cabeza（Caz wt，P398 L，Q349 X）的相应变体。这些变体在眼睛和运动神经元中的表达证实了FUS/Caz的PY-NLS依赖性核定位并引起神经退行性作用。令人惊讶的是，FUS/Caz毒性与其在该过表达模型中的核定位程度相关。高水平的核FUS/Caz变得不可溶，降低了内源性Caz水平，证实了果蝇中的PUS自动调节。RNAi介导的两种转运蛋白同源物的敲除干扰了FUS/Caz的核输入，并且还增强了眼表型。最后，我们对果蝇PRMT蛋白（DART 1 -9）进行了筛选，发现Dart 1的敲低导致hFUS P525 L甲基化水平降低，并使其表型恶化，表明控制FUS/Caz核内输入和FUS自身调节的分子机制在人和果蝇之间是保守的。除了众所周知的FUS功能丧失的神经退行性作用外，我们的数据表明细胞核中过表达的FUS和不溶性PUS的毒性潜力。(C)2014爱思唯尔公司All rights reserved.
Inclusions containing Fused in Sarcoma (PUS) are found in familial and sporadic cases of the incurable progressive motor neuron disease amyotrophic lateral sclerosis and in a common form of dementia, frontotemporal dementia. Most disease-associated mutations are located in the C-terminal proline-tyrosine nuclear localization sequence (PY-NLS) of PUS and impair its nuclear import. It has been shown in cell culture that the nuclear import of PUS is mediated by transportin, which binds the PY-NLS and the last arginine/glyc-ine/glycine-rich (RGG) domain of FUS. Methylation of this last RGG domain by protein arginine methyltransferases (PRMTs) weakens transportin binding and therefore impairs nuclear translocation of FUS. To investigate the requirements for the nuclear import of PUS in an in vivo model, we generated different transgenic Drosophila lines expressing human FUS wild type (hFUS wt) and two disease-related variants P525L and R495X, in which the NLS is mutated or completely absent, respectively. To rule out effects caused by heterologous hFUS expression, we analysed the corresponding variants for the Drosophila FUS orthologue Cabeza (Caz wt, P398L, Q349X). Expression of these variants in eyes and motor neurons confirmed the PY-NLS-dependent nuclear localization of FUS/Caz and caused neurodegenerative effects. Surprisingly, FUS/Caz toxicity was correlated to the degree of its nuclear localization in this overexpression model. High levels of nuclear FUS/Caz became insoluble and reduced the endogenous Caz levels, confirming PUS autoregulation in Drosophila. RNAi-mediated knockdown of the two transportin orthologues interfered with the nuclear import of FUS/Caz and also enhanced the eye phenotype. Finally, we screened the Drosophila PRMT proteins (DART1-9) and found that knockdown of Dart] led to a reduction in methylation of hFUS P525L and aggravated its phenotype.These findings show that the molecular mechanisms controlling the nuclear import of FUS/Caz and FUS autoregulation are conserved between humans and Drosophila. In addition to the well-known neurodegenerative effects of FUS loss-of function, our data suggest toxic potential of overexpressed FUS in the nucleus and of insoluble PUS. (C) 2014 Elsevier Inc. All rights reserved.