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Studies on the ion channel activity of influenza C virus CM2 protein

丙型流感病毒CM2蛋白离子通道活性研究

基本信息

批准号：
11670287
负责人：
HONGO Seiji
金额：
$ 2.43万
依托单位：
YAMAGATA UNIVERSITY
依托单位国家：
日本
项目类别：
Grant-in-Aid for Scientific Research (C)
财政年份：
1999
资助国家：
日本
起止时间：
1999 至 2000
项目状态：
已结题

来源：
https://kaken.nii.ac.jp/grant/KAKENHI-PROJECT-11670287/
关键词：
Influenza C virus CM2 posttranslational modification ion channel

项目摘要

The sites for fatty acylation, disulfide bond formation and phosphorylation of influenza C virus CM2 were investigated by site-specific mutagenesis. Cysteine 65 in the cytoplasmic tail was identified as the site for palmitoylation. Removal of one or more of three cysteine residues in the ectodomain showed that all of cysteines 1, 6, and 20 can participate in the formation of disulfide- linked dimers and/or tetramers, although cysteine 20 may play the most important role in tetramer formation. Furthermore, it was found that serine 78, located within the recognition motifs for mammary gland casein kinase and casein kinase I, is the predominant site for phosphorylation, although serine 103 is phosphorylated to a minor extent by proline -dependent protein kinase. The effects of acylation and phosphorylation on the formation of disulfide-linked oligomers were also studied. The results showed that, while palmitoylation has no role in oligomer formation, phosphorylation accelerates tetramer formation without influencing dimer formation. CM2 mutants defective in acylation, phosphorylation or disulfide bond formation were all transported to the cell surface, suggesting that none of these modifications is required for proper oligomerization. When proteins solubilized in detergent were analysed on sucrose gradients, however, the mutant lacking cysteines 1, 6 and 20 sedimented as monomers, raising the possibility that disulfide bond formation, although not essential for proper oligomerization, may stabilize the CM2 multimer. This was supported by the results of chemical cross-linking analysis which showed that the triple cysteine mutant can form multimers.

用定点突变的方法研究了C型流感病毒CM2的脂肪酰化、二硫键形成和磷酸化的位点。胞质尾部的半胱氨酸65被确定为棕榈酰化的位点。胞外结构域中三个半胱氨酸残基中的一个或多个的去除表明，所有的半胱氨酸1、6和20都可以参与二硫键连接的二聚体和/或四聚体的形成，尽管半胱氨酸20可能在四聚体的形成中发挥最重要的作用。此外，研究还发现，位于乳腺酪蛋白激酶和酪蛋白激酶I识别基序中的丝氨酸78是主要的磷酸化位点，尽管丝氨酸103被脯氨酸依赖的蛋白激酶有少量的磷酸化。还研究了酰化和磷酸化对二硫键低聚物形成的影响。结果表明，棕榈酰化对低聚物的形成没有影响，而磷酸化促进了四聚体的形成，而不影响二聚体的形成。在酰化、磷酸化或二硫键形成方面存在缺陷的CM2突变体都被运输到细胞表面，表明这些修饰都不是适当的寡聚所必需的。然而，当对溶解在洗涤剂中的蛋白质进行蔗糖梯度分析时，缺乏半胱氨酸1、6和20的突变体以单体形式沉淀，这增加了二硫键形成的可能性，尽管二硫键的形成对适当的齐聚不是必需的，但可能稳定CM2多聚体。化学交联分析结果表明，三重半胱氨酸突变体可以形成多聚体。