Formation of morphologically similar globular aggregates from diverse aggregation-prone proteins in mammalian cells.
Formation of morphologically similar globular aggregates from diverse aggregation-prone proteins in mammalian cells.
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DOI：
10.1073/pnas.0409283102
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发表时间：
2005-08
影响因子：
11.1
通讯作者：
H. Mukai;T. Isagawa;Emiko Goyama;Shuhei Tanaka;N. Bence;A. Tamura;Y. Ono;R. Kopito
中科院分区：
文献类型：
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作者：
H. Mukai;T. Isagawa;Emiko Goyama;Shuhei Tanaka;N. Bence;A. Tamura;Y. Ono;R. Kopito
亨廷顿氏病是一种进行性神经退行性疾病，由亨廷顿蛋白（Htt）第一外显子的多聚谷氨酰胺重复扩增引起。具有病理范围（51-122谷氨酰胺）的聚谷氨酰胺束的n端Htt肽在体外形成具有纤维状形态的高分子量蛋白质聚集体，并在细胞培养模型中形成离散包涵体。然而，在一些研究中，离散Htt包涵体的形成与细胞死亡没有很好的相关性。我们在HEK293细胞中共表达含有91个谷氨酰胺融合到不同亲和标签的n端Htt片段，并通过双序列亲和层析分离小聚集体以确保多聚分子的分离。透射电子显微镜和原子力显微镜显示，分离的聚集体为直径4-50 nm的球状或球状团，没有任何可检测到的纤维物质。由于小的非原纤维低聚物，而不是成熟的原纤维，最近被认为是神经退行性疾病的主要细胞毒性物质，这些在细胞中形成的Htt球状聚集体可能代表突变Htt的致病形式。
Huntington's disease is a progressive neurodegenerative disorder caused by a polyglutamine repeat expansion in the first exon of the huntingtin (Htt) protein. N-terminal Htt peptides with polyglutamine tracts in the pathological range (51-122 glutamines) form high-molecular-weight protein aggregates with fibrillar morphology in vitro, and they form discrete inclusion bodies in a cell-culture model. However, in some studies, formation of discrete Htt inclusions does not correlate well with cell death. We coexpressed N-terminal Htt fragments containing 91 glutamines fused to different affinity tags in HEK293 cells, and we isolated small aggregates by double sequential-affinity chromatography to assure the isolation of multimeric molecules. Transmission electron microscopy and atomic force microscopy revealed the isolated aggregates as globules or clusters of globules 4-50 nm in diameter without any detectable fibrillar species. Because small nonfibrillar oligomers, not mature fibrils, recently have been suggested to be the principal cytotoxic species in neurodegenerative disease, these Htt globular aggregates formed in cells may represent the pathogenic form of mutant Htt.