In Vitro and in Vivo Aggregation of a Fragment of Huntingtin Protein Directly Causes Free Radical Production
In Vitro and in Vivo Aggregation of a Fragment of Huntingtin Protein Directly Causes Free Radical Production
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DOI：
10.1074/jbc.m111.307587
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发表时间：
2011-12-30
影响因子：
4.8
通讯作者：
Wyttenbach, Andreas
中科院分区：
文献类型：
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作者：
Hands, Sarah;Sajjad, Mohammad U.;Wyttenbach, Andreas
神经退行性疾病的特征在于细胞内和/或细胞外蛋白质聚集和氧化应激。蛋白质聚集本身是否会导致自由基的异常产生和随后的细胞氧化损伤一直受到人们的密切关注。虽然这个问题已经在细胞外蛋白质聚集的背景下进行了研究，但仍不清楚细胞内的蛋白质聚集是否会改变氧化还原稳态。为了解决这一问题，我们使用了亨廷顿病（九种多聚谷氨酰胺（poly（Q））疾病之一）的体外和体内（细胞）模型，并研究了细胞内蛋白质聚集、活性氧（ROS）产生和毒性之间的因果关系。用poly（Q）扩增表达亨廷顿蛋白片段（httpExon 1）的细胞的实时成像显示细胞死亡前ROS产生增加。ROS的产生是聚（Q）长度依赖性的，而不是由于httpExon 1侧翼序列。通过MW 7胞内抗体和Pgl-135处理的聚集抑制消除了ROS产生，表明增加的ROS是由聚（Q）聚集本身引起的。为了进一步检验这一假设，我们确定了聚（Q）肽在体外的聚集是否产生自由基。使用原子力显微镜和过氧化氢（H2 O2）的生产随着时间的推移并行监测聚（Q）蛋白质聚集，我们表明，寡聚化的HTTEx 1 Q53的结果在早期生成的H2 O2。单链抗体MW 7对聚（Q）寡聚化的抑制消除了H2 O2的形成。这些结果表明，细胞内蛋白质聚集直接导致自由基的产生，靶向潜在的毒性聚（Q）寡聚体可能构成一个治疗目标，以抵消聚（Q）疾病中的氧化应激。
Neurodegenerative diseases are characterized by intra- and/or extracellular protein aggregation and oxidative stress. Intense attention has been paid to whether protein aggregation itself contributes to abnormal production of free radicals and ensuing cellular oxidative damage. Although this question has been investigated in the context of extracellular protein aggregation, it remains unclear whether protein aggregation inside cells alters the redox homeostasis. To address this, we have used in vitro and in vivo (cellular) models of Huntington disease, one of nine polyglutamine (poly(Q)) disorders, and examined the causal relationship among intracellular protein aggregation, reactive oxygen species (ROS) production, and toxicity. Live imaging of cells expressing a fragment of huntingtin (httExon1) with a poly(Q) expansion shows increased ROS production preceding cell death. ROS production is poly(Q) length-dependent and not due to the httExon 1 flanking sequence. Aggregation inhibition by the MW7 intrabody and Pgl-135 treatment abolishes ROS production, showing that increased ROS is caused by poly(Q) aggregation itself. To examine this hypothesis further, we determined whether aggregation of poly(Q) peptides in vitro generated free radicals. Monitoring poly(Q) protein aggregation using atomic force microscopy and hydrogen peroxide (H2O2) production over time in parallel we show that oligomerization of httEx1Q53 results in early generation of H2O2. Inhibition of poly(Q) oligomerization by the single chain antibody MW7 abrogates H2O2 formation. These results demonstrate that intracellular protein aggregation directly causes free radical production, and targeting potentially toxic poly(Q) oligomers may constitute a therapeutic target to counteract oxidative stress in poly(Q) diseases.