Two Conserved Cysteine Residues Are Required for the Masculinizing Activity of the Silkworm Masc Protein
Two Conserved Cysteine Residues Are Required for the Masculinizing Activity of the Silkworm Masc Protein
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DOI：
10.1074/jbc.m115.685362
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发表时间：
2015-10-23
影响因子：
4.8
通讯作者：
Shimada, Toru
中科院分区：
文献类型：
--
作者：
Katsuma, Susumu;Sugano, Yudai;Shimada, Toru
我们最近发现，Masc基因编码一个CCCH串联锌指蛋白，它控制着家蚕的雄性化和剂量补偿。在这项研究中，我们试图确定Masc蛋白男性化活性所需的功能区或残基。我们构建了一系列表达Masc衍生物的质粒，并将它们导入家蚕卵巢来源的细胞系BMN-4。为了评估Masc衍生物的男性化活性，我们研究了双重家蚕(B.mori doublesex，Bmdsx)的剪接模式以及Bmdsx的剪接调节因子B.mori IGF-II mRNA结合蛋白在Masc cDNA转染的BMN-4细胞中的表达水平。我们发现，男性化活性不需要两个锌指结构域。我们还鉴定了C端的288个氨基酸残基足以满足Masc蛋白的雄性化活性。进一步的详细分析表明，鳞翅目昆虫Masc蛋白高度保守区的两个半胱氨酸残基Cys-301和Cys-304对BmN-4细胞的雄性化活性是必不可少的。最后，我们证明了Masc是一种核蛋白，但它的核定位与男性化活性并不密切相关。
We have recently discovered that the Masculinizer (Masc) gene encodes a CCCH tandem zinc finger protein, which controls both masculinization and dosage compensation in the silkworm Bombyx mori. In this study, we attempted to identify functional regions or residues that are required for the masculinizing activity of the Masc protein. We constructed a series of plasmids that expressed the Masc derivatives and transfected them into a B. mori ovary-derived cell line, BmN-4. To assess the masculinizing activity of the Masc derivatives, we investigated the splicing patterns of B. mori doublesex (Bmdsx) and the expression levels of B. mori IGF-II mRNA-binding protein, a splicing regulator of Bmdsx, in Masc cDNA-transfected BmN-4 cells. We found that two zinc finger domains are not required for the masculinizing activity. We also identified that the C-terminal 288 amino acid residues are sufficient for the masculinizing activity of the Masc protein. Further detailed analyses revealed that two cysteine residues, Cys-301 and Cys-304, in the highly conserved region among lepidopteran Masc proteins are essential for the masculinizing activity in BmN-4 cells. Finally, we showed that Masc is a nuclear protein, but its nuclear localization is not tightly associated with the masculinizing activity.