Ataxin-1 Fusion Partners Alter PolyQ Lethality and Aggregation
Ataxin-1 Fusion Partners Alter PolyQ Lethality and Aggregation
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DOI：
10.1371/journal.pone.0001014
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发表时间：
2007-10-10
期刊：
影响因子：
3.7
通讯作者：
Varadaraj, Archana
中科院分区：
文献类型：
--
作者：
Rich, Tina;Varadaraj, Archana
核内包涵体是多种蛋白质折叠疾病的组织病理学标志。IB的形成已被广泛研究使用荧光融合产物的致病性多聚谷氨酰胺（polyQ）表达蛋白。这些研究在确定扩增的polyQ蛋白的细胞靶点以及细胞摆脱IB的方法方面提供了信息。实验的重点是干预polyQ聚集的过程，试图减轻细胞毒性。然而，新的数据反驳了polyQ聚集和细胞毒性是不可分割的联系过程的观点。我们推断，改变引起疾病的polyQ蛋白的蛋白质背景可以加速其作为IB的沉淀，从而潜在地降低其细胞毒性。我们的实验策略简单地利用了这样一个事实，即连接的蛋白质会影响彼此的折叠和聚集特性。我们将全长致病性共济失调蛋白-1构建体融合到以不同寡聚状态存在的荧光标签（GFP和DsRed 1-E5）。DsRed 1-E5-共济失调蛋白-1转染子的光谱特性具有使我们能够将荧光染料成熟与细胞毒性相关联的额外优点。每种融合蛋白表达不同的细胞毒性和IB形态。表达最大荧光信号的转染子的流式细胞术分析显示，DsRed 1-E5-共济失调蛋白-1融合体比GFP融合的共济失调蛋白-1毒性更大（31.8+/-4.5%细胞死亡对12.85+/-3%），尽管与GFP融合体共转染抑制了DsRed 1-E5荧光染料的成熟并降低了DsRed 1-E5-共济失调蛋白-1融合体的毒性。这些数据表明，polyQ驱动的聚集可以受到融合伴侣的影响，以产生具有不同毒性特性的物种，并为研究IB聚集、成熟和致死性提供了新的机会。
Intranuclear inclusion bodies (IBs) are the histopathologic markers of multiple protein folding diseases. IB formation has been extensively studied using fluorescent fusion products of pathogenic polyglutamine (polyQ) expressing proteins. These studies have been informative in determining the cellular targets of expanded polyQ protein as well as the methods by which cells rid themselves of IBs. The experimental thrust has been to intervene in the process of polyQ aggregation in an attempt to alleviate cytotoxicity. However new data argues against the notion that polyQ aggregation and cytotoxicity are inextricably linked processes. We reasoned that changing the protein context of a disease causing polyQ protein could accelerate its precipitation as an IB, potentially reducing its cytotoxicity. Our experimental strategy simply exploited the fact that conjoined proteins influence each others folding and aggregation properties. We fused a full-length pathogenic ataxin-1 construct to fluorescent tags (GFP and DsRed1-E5) that exist at different oligomeric states. The spectral properties of the DsRed1-E5-ataxin-1 transfectants had the additional advantage of allowing us to correlate fluorochrome maturation with cytotoxicity. Each fusion protein expressed a distinct cytotoxicity and IB morphology. Flow cytometric analyses of transfectants expressing the greatest fluorescent signals revealed that the DsRed1-E5-ataxin-1 fusion was more toxic than GFP fused ataxin-1 (31.8+/-4.5% cell death versus 12.85+/-3%), although co-transfection with the GFP fusion inhibited maturation of the DsRed1-E5 fluorochrome and diminished the toxicity of the DsRed1-E5-ataxin-1 fusion. These data show that polyQ driven aggregation can be influenced by fusion partners to generate species with different toxic properties and provide new opportunities to study IB aggregation, maturation and lethality.