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.

通过定点诱变研究了丙型流感病毒CM2的脂肪酰化、二硫键形成和磷酸化位点。细胞质尾部的半胱氨酸 65 被确定为棕榈酰化位点。胞外域中三个半胱氨酸残基中的一个或多个的去除表明，所有半胱氨酸 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 可能在四聚体形成中发挥最重要的作用。此外，发现位于乳腺酪蛋白激酶和酪蛋白激酶I的识别基序内的丝氨酸78是磷酸化的主要位点，尽管丝氨酸103被脯氨酸依赖性蛋白激酶小程度地磷酸化。还研究了酰化和磷酸化对二硫键连接的低聚物形成的影响。结果表明，虽然棕榈酰化对寡聚体的形成没有作用，但磷酸化可加速四聚体的形成而不影响二聚体的形成。酰化、磷酸化或二硫键形成缺陷的 CM2 突变体均被转运至细胞表面，表明适当的寡聚化不需要这些修饰。然而，当在蔗糖梯度上分析溶解在去污剂中的蛋白质时，缺乏半胱氨酸 1、6 和 20 的突变体作为单体沉淀，这提高了二硫键形成的可能性，尽管对于适当的寡聚化不是必需的，但可能稳定 CM2 多聚体。化学交联分析结果支持了这一点，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。