Toxicity and endocytosis of spinocerebellar ataxia type 6 polyglutamine domains:: Role of myosin IIB
Toxicity and endocytosis of spinocerebellar ataxia type 6 polyglutamine domains:: Role of myosin IIB
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DOI：
10.1111/j.1600-0854.2008.00743.x
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发表时间：
2008-07-01
期刊：
影响因子：
4.5
通讯作者：
Seagar, Michael
中科院分区：
文献类型：
--
作者：
Marqueze-Pouey, Beatrice;Martin-Moutot, Nicole;Seagar, Michael
脊髓小脑共济失调 6 型 (SCA6) 是一种显性遗传性神经退行性疾病，由编码 P/Q 型钙通道 Ca(v)2.1 亚基细胞质 C 端区域的序列中 CAG 重复的小幅扩展引起。我们测试了质膜中表达的突变 Ca(v)2.1 C 端结构域的毒性。在 COS-7 细胞中，与“正常”12 个 Q 束相比，与含有扩展的 24 个聚谷氨酰胺 (Q) 束的 Ca(v)2.1 C 端结构域融合的 CD4 绿色荧光蛋白在细胞表面表现出更高的毒性和更强的表达，部分原因是内吞作用减少。谷胱甘肽 S-转移酶下拉和蛋白质组分析表明 Ca(v)2.1 C 末端与小脑肌球蛋白 IIB（一种分子运动蛋白）的重链和轻链相互作用。这种相互作用通过大鼠小脑和 COS-7 细胞的免疫共沉淀得到证实，并且表明是通过体外翻译的 S-35-肌球蛋白 IIB 重链的结合直接实现的。在 COS-7 细胞中，增加的聚谷氨酰胺束长度增加了与肌球蛋白 IIB 的相互作用。此外，肌球蛋白 II 抑制剂 blebbistatin 逆转了多聚谷氨酰胺扩增对质膜表达的影响。我们的研究结果表明肌球蛋白 IIB 在促进突变型 Ca(v)2.1Ct 在质膜上积累方面发挥着关键作用，并表明这种功能的获得可能有助于 SCA6 的发病机制。
Spinocerebellar ataxia type 6 (SCA6) is a dominantly inherited neurodegenerative disease caused by a small expansion of CAG repeats in the sequence coding for the cytoplasmic C-terminal region of the Ca(v)2.1 subunit of P/Q-type calcium channels. We have tested the toxicity of mutated Ca(v)2.1 C-terminal domains expressed in the plasma membrane. In COS-7 cells, CD4-green fluorescent protein fused to Ca(v)2.1 C-terminal domains containing expanded 24 polyglutamine (Q) tracts displayed increased toxicity and stronger expression at the cell surface relative to 'normal' 12 Q tracts, partially because of reduced endocytosis. Glutathione S-transferase pull-down and proteomic analysis indicated that Ca(v)2.1 C-termini interact with the heavy and light chains of cerebellar myosin IIB, a molecular motor protein. This interaction was confirmed by coimmunoprecipitation from rat cerebellum and COS-7 cells and shown to be direct by binding of in vitro-translated S-35-myosin IIB heavy chain. In COS-7 cells, incremented polyglutamine tract length increased the interaction with myosin IIB. Furthermore, the myosin II inhibitor blebbistatin reversed the effects of polyglutamine expansion on plasma membrane expression. Our findings suggest a key role of myosin IIB in promoting accumulation of mutant Ca(v)2.1Ct at the plasma membrane and suggest that this gain of function might contribute to the pathogenesis of SCA6.