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Studies on the controlling mechanism of endocrine cell proliferation mediated by the MEN1 gene product menin

MEN1基因产物menin介导的内分泌细胞增殖调控机制研究

基本信息

批准号：
17590283
负责人：
TSUKADA Toshihiko
金额：
$ 2.43万
依托单位：
National Cancer Center Research Institute and Research Center for Innovative Oncology, National Cancer Center Hospital East
依托单位国家：
日本
项目类别：
Grant-in-Aid for Scientific Research (C)
财政年份：
2005
资助国家：
日本
起止时间：
2005 至 2007
项目状态：
已结题

项目摘要

1. To elucidate the controlling mechanism for endocrine cell proliferation mediated by menin, the protein product of the causative gene for multiple endocrine neoplasia type 1, proteins binding to menin in endocrine cells were explored by immunoprecipitation. Insulin-secreting culture cells derived from a rat pancreatic islet tumor were stably transfected with plasmid expressing Flag-tagged menin. Immunoprecipitation of the cell lysate with anti-Flag antibody yeilded several proteins coprecipitated with menin. Mass spectrometric analyses revealed that the coprecipitated proteins included heat shock proteins, ribosomal proteins and transcription factor-like proteins. The latter has been shown to bind weakly to menin by the mammalian cell two-hybrid analysis. Immunocytochemical analyses demonstrated that this menin-binding protein is localized to juxtanuclear area in a spotted pattern.2. Menin has previously been demonstrated to have GTPase activity. Menin synthesized by the in vitro transcription and translation system was tested for GTPase activity. The in vitrosynthesized menin failed to show GTPase activity, suggesting that the post-translational modification may be required to have the enzyme activity of menin.3. By the experiments with parathyroid tumor cells derived from a patient of multiple endocrine neoplasia type 1, menin has been shown to mediate cell growth-inhibiting effects of TGF-β on parathyroid cells.4. Immunoblotting analyses of various missense mutant menin proteins that are forced to be expressed in culture cells demonstrated that almost all pathogenic missense MEN1 gene mutations caused rapid degradation of menin. These findings suggest that instability of the mutant menin is involved in the pathogenesis of multiple endocrine neoplasia type 1 caused by the missense mutations of the MEN1 gene.

1.为阐明多发性内分泌腺瘤1型（multiple endocrine neoplasia type 1）致病基因的蛋白产物menin对内分泌细胞增殖的调控机制，采用免疫沉淀法研究了内分泌细胞中与menin结合的蛋白。用表达Flag标记的menin的质粒稳定转染来自大鼠胰岛肿瘤的胰岛素分泌培养细胞。用抗Flag抗体免疫沉淀细胞裂解物，产生几种与menin共沉淀的蛋白质。质谱分析表明，共沉淀蛋白包括热休克蛋白、核糖体蛋白和转录因子样蛋白。通过哺乳动物细胞双杂交分析，后者已被证明与menin弱结合。免疫细胞化学分析表明，该menin结合蛋白定位于细胞核内，呈斑点状. Menin先前已被证明具有GT3活性。测试通过体外转录和翻译系统合成的Menin的GT3活性。体外合成的menin没有表现出GT3活性，提示menin可能需要翻译后修饰才具有酶活性.通过对1型多发性内分泌瘤患者的甲状旁腺肿瘤细胞的实验，menin可介导TGF-β对甲状旁腺细胞的细胞生长抑制作用.被迫在培养细胞中表达的各种错义突变menin蛋白的免疫印迹分析表明，几乎所有致病性错义MEN 1基因突变引起menin的快速降解。这些结果表明，突变的menin的不稳定性参与多发性内分泌腺瘤1型的发病机制所造成的MEN 1基因的错义突变。