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Metabolic regulation of estrogen-synthesizing enzymes by post-translational modification

翻译后修饰对雌激素合成酶的代谢调节

基本信息

批准号：
18591034
负责人：
HARADA Nobuhiro
金额：
$ 2.57万
依托单位：
Fujita Health University
依托单位国家：
日本
项目类别：
Grant-in-Aid for Scientific Research (C)
财政年份：
2006
资助国家：
日本
起止时间：
2006 至 2007
项目状态：
已结题

项目摘要

In this study project, we focussed on the post-translational modification of aromatase, a rate-limiting enzyme of estrogen production, and analyzed molecular and regulatory mechanisms of the post-translational modification and physiological roles of this reaction. Western blotting analysis showed a possibility of post-translational modification of aromatase by phosphorylation in the cultured cells in the presence of various inducers and regulatory factors. Furthermore, ELISA assay of aromatase contents in the cultured cells suggested a possiblity that the intracellular degradation rate (the turnover rate) was modified by various factors. So, The in vitro assay system was introduced to analyze the process of the intracelluar degradation of the aromatase in the endoplasmic reticulum (microsomal fraction). The degradation of the microsomal aromatase content and activity required the presence of ATP-Mg2+ and the cytoplasmic fraction in the reaction. An aromatase antibody-immunoreactive protein with a high molecular weight (AR-ir protein) appeared in parallel with disappearance of the authentic aromatase with the molecular weight of 53, 000 by Western blotting analysis. The disappearance of the aromatase was inhibited by presence of Geldanamycin, an inhibitor of HSP90 chaperone protein. However, because the purified recombinant aromatase did not disappear and the AR-ir protein did not appear in the assay and also because the microsomal aromatase did not disappear when low concentration of cholic acid was added to the assay system to solubilize the microsomal membrane, it was suggested that the microsomal aromatase was quantitatively regulated by the microsomal membrane-dependent degradation system together with cytoplasmic regulatory proteins. We are now analyzing the constitutive factors participating in the microsomal membrane-dependent degradation by using FLAG-tagged aromatase.

在本研究项目中，我们重点关注雌激素产生限速酶芳香酶的翻译后修饰，并分析了翻译后修饰的分子和调控机制以及该反应的生理作用。蛋白质印迹分析表明，在各种诱导剂和调节因子存在的情况下，培养细胞中芳香酶可能通过磷酸化进行翻译后修饰。此外，对培养细胞中芳香酶含量的ELISA测定表明，细胞内降解率（周转率）可能受到多种因素的影响。因此，引入体外测定系统来分析芳香酶在内质网（微粒体部分）的细胞内降解过程。微粒体芳香酶含量和活性的降解需要反应中存在 ATP-Mg2+ 和细胞质部分。通过蛋白质印迹分析，在分子量为 53, 000 的真实芳香酶消失的同时，出现了高分子量芳香酶抗体免疫反应蛋白（AR-ir 蛋白）。芳香酶的消失受到格尔德霉素（HSP90 伴侣蛋白抑制剂）的存在的抑制。然而，由于纯化的重组芳香酶没有消失，且AR-ir蛋白没有出现在测定中，而且当向测定系统中添加低浓度胆酸溶解微粒体膜时微粒体芳香酶也没有消失，因此表明微粒体芳香酶受到微粒体膜依赖性降解系统与细胞质的定量调节。调节蛋白。我们现在正在使用 FLAG 标记的芳香酶分析参与微粒体膜依赖性降解的组成因素。