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

在本研究项目中，我们重点研究了雌激素生成限速酶芳香化酶的翻译后修饰，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。Western blotting分析显示，在多种诱导剂和调节因子存在的情况下，培养细胞中存在磷酸化修饰芳香化酶的可能性。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）可能受到各种因素的影响。为此，本文引入体外检测系统，分析内质网（微粒体部分）中芳香化酶的细胞内降解过程。微粒体芳香酶含量和活性的降解需要ATP-Mg2+和细胞质部分的存在。Western blotting分析显示，高分子量的芳香化酶抗体免疫反应蛋白（AR-ir蛋白）与分子量为53,000的真正芳香化酶同时消失。格尔达霉素（一种HSP90伴侣蛋白抑制剂）的存在抑制了芳香酶的消失。然而，由于纯化后的重组芳香化酶没有消失，AR-ir蛋白也没有出现在检测中，并且当添加低浓度胆酸溶解微粒体膜时，微粒体芳香化酶也没有消失，提示微粒体芳香化酶与细胞质调节蛋白一起受到微粒体膜依赖性降解系统的定量调节。我们现在通过使用flag标记的芳香酶分析参与微粒体膜依赖性降解的构成因素。

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.